Serling was born on December 25, 1924, in Syracuse, New York, to a Jewish family. He was the second of two sons born to Esther (nee Cooper) and Samuel Lawrence Serling. Serling's father had worked as a secretary and amateur inventor before having children, but took on his father-in-law's profession as a grocer to earn a steady income. Sam Serling later became a butcher after the Great Depression forced the store to close.

After being discharged from the Army in 1946, Serling worked at a rehabilitation hospital while recovering from his wounds. His knee troubled him for years. Later, his wife became accustomed to the sound of him falling down the stairs when his knee buckled under his weight.  When he was fit enough, he used the federal G.I. bill's educational benefits and disability payments to enroll in the physical education program at Antioch College in Yellow Springs, Ohio. He had been accepted to Antioch (his brother's alma mater) while in high school. His interests led him to the theater department and then to broadcasting. He changed his major to Literature and earned his Bachelor of Arts degree in 1950. "I was kind of mixed up and restless, and I kind of liked their work-for-a-term, go-to-school-for-a-term set-up," he recounted.  As part of his studies, Serling became active in the campus radio station, an experience which proved useful in his future career. He wrote, directed, and acted in many radio programs on campus, then around the state, as part of his work study. Here he met Carolyn Louise "Carol" Kramer, a student, who later became his wife. At first, she refused to date him because of his campus reputation as a "ladies man", but she eventually changed her mind. He converted from Judaism to Unitarianism in college, which allowed him to marry Kramer on July 31, 1948. They had two daughters, Jodi and Anne.  Carol Serling's maternal grandmother, Louise Taft Orton Caldwell, had a summer home on Cayuga Lake in Interlaken, New York, which was the newlyweds' honeymoon destination. The Serling family continued to use this house annually throughout Rod's life, missing only two summers in the years when his daughters were born.  For extra money in his college years, Serling worked part-time testing parachutes for the United States Army Air Forces. According to his radio station coworkers, he received $50 for each successful jump and had once been paid $500 (half before and half if he survived) for a hazardous test. His last test jump was a few weeks before his wedding. In one instance, he earned $1,000 for testing a jet ejection seat that had killed the previous three testers.

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