Stephanie Louise Kwolek was born in New Kensington, Pennsylvania, to Polish immigrant parents, John Kwolek (a factory worker) and Nellie (Zajdel) Kwolek (a homemaker).

Kwolek developed an early fascination with science, inspired by her father's interest in nature and her mother's skills in sewing, which sparked her curiosity about fabrics and textiles. She enjoyed spending time outdoors and exploring nature.

Stephanie's father passed away, influencing her to take part time jobs and take on responsibilities to help support the family.

Kwolek graduated with a Bachelor of Science degree in Chemistry from Margaret Morrison Carnegie College of Carnegie Mellon University. Her original intention was to become a doctor, and she hoped to earn enough money working as a chemist to attend medical school.