Biscayne Bay is a lagoon with characteristics of an estuary located on the Atlantic coast of South Florida. The northern end of the lagoon is surrounded by the densely developed heart of the Miami metropolitan area while the southern end is largely undeveloped with a large portion of the lagoon included in Biscayne National Park.

The part of the lagoon that is traditionally called "Biscayne Bay" is approximately 35 miles (56 km) long and up to 8 miles (13 km) wide, with a surface area of 221 square miles (570 km²). Various definitions may include Dumfoundling Bay, Card Sound, and Barnes Sound in a larger "Biscayne Bay", which is 60 miles (97 km) long with a surface area of about 271 square miles (700 km²).

Hernando de Escalante Fontaneda related in the 16th century that a sailor from the Bay of Biscay called the Viscayno or Biscayno had lived on the lower east coast of Florida for a while after being shipwrecked, and a 17th-century map shows a Cayo de Biscainhos, the probable origin of the name for Key Biscayne. The lagoon was known as "Key Biscayne Bay" in the 19th century, finally shrinking to "Biscayne Bay" late in the 19th century.

The lagoon has been known by several names. Juan Ponce de León called it Chequescha in 1513, and Pedro Menéndez de Avilés called it Tequesta in 1565. Those names are variant spellings of "Tequesta", the name of the people who lived around the lagoon at the time. The British, during their occupation of Florida (1763–1783), called the lagoon "Cape River", "Dartmouth Sound", and "Sandwich gulph".

Biscayne Bay is a semi- or subtropical lagoon extending most of the length of Miami-Dade County, from North Miami Beach to the upper Florida Keys. Biscayne Bay, in the strictest sense, extends from a point between North Miami Beach and Sunny Isles Beach south to the Arsenicker Keys and the Cutter Bank just to the east of those islands. Many discussions about the lagoon include Dumfoundling Bay, a small lagoon just north of North Miami Beach, as part of Biscayne Bay, and include Card Sound and Barnes Sound, in southern Miami-Dade County adjacent to Key Largo, as either part of a system of connected lagoons including Biscayne Bay, or as part of Biscayne Bay itself. The lagoon is bordered on its west by the mainland of Florida, and is separated from the Atlantic Ocean to the east by a string of barrier islands in the northern part of the lagoon, a large shoal in the central section, and the northernmost of the Florida Keys in the south. The lagoon is about 35 miles (56 km) long from Dumfoundling Bay (25° 58′ North latitude) (north of North Miami Beach) to Card Sound, and another 25 miles (40 km) to Jewfish Creek (25° 24′ North latitude), if Card Sound and Barnes Sound are included in the lagoon. The lagoon is 8 miles (13 km) across at its widest point. The lagoon, from Dumfoundling Bay to the Arsenicker Keys, has a surface area of about 572 square kilometres (221 sq mi). The larger lagoon, including Card Sound and Barnes Sound, has a surface area of about 703 square kilometres (271 sq mi). It has an average depth of 1.8 metres (5.9 ft) and, except where channels have been dredged, a maximum depth of 4 metres (13 ft).

The eastern rim of the lagoon is based on an ancient coral reef which existed along the southeastern edge of the Florida Platform about 100,000 years ago. The ensuing Wisconsin glaciation lowered sea levels, leaving the reef above water. The dead reef became fossilized, forming the Key Largo Limestone (commonly called "coral rock"). Key Largo Limestone underlies the Eastern edge of the barrier islands and the shoals (Safety Valve) along the northern and middle part of the lagoon. It makes a brief appearance at Soldier Key, in the middle of the Safety Valve, and rises above sea level to the south to form the upper Florida Keys.

The western side of the bay has characteristics of an estuary, with 16 waterways, the largest of which is the Miami River, flowing into it. All of the waterways have saltwater intrusion control structures, which restrict the flow of saltwater inland, and regulate the flow of fresh water into the lagoon. Some fresh water enters the lagoon as groundwater flow from the Biscayne Aquifer. All of the bedrock under the lagoon west of the ancient reef, and the adjacent mainland, consists of Miami Limestone, an oolitic limestone that formed in a lagoon behind the ancient reef that became the Key Largo limestone, and is the same age as the Key Largo limestone. The limestone bottom of the lagoon is overlain by 1.2 to 4.6 metres (3.9 to 15.1 ft) of sand, carbonate mud, and coral rubble sediments.

For purposes of discussion and analysis, Biscayne Bay is often divided into three sections: North Bay, Central Bay, and South Bay.