Tyus, from Tennessee State University, participated in the 1964 Summer Olympics at age 19. In the heats of the event, she equaled Wilma Rudolph's world record, propelling her to a favored position for the final, where her main rival was fellow American Edith McGuire. Tyus won the final, beating McGuire by 0.2 seconds. At the same Olympics, she also won a silver medal with the 4×100 m relay team.
The following years, Tyus won numerous national championships in the sprint events, and a gold medal in the 200 m at the Pan-American Games. In 1968, she returned to the Olympics to defend her title in the 100 m. In the final, she set a new world record of 11.08 s to become the first woman to retain the Olympic 100 m title. Tyus also qualified for the 200 m final, in which she finished sixth. Running the final leg for the relay team, Tyus helped setting a new world record, winning her third gold medal.
Director Bud Greenspan filmed Tyus casually dancing behind her starting blocks before the Olympic final. When interviewed later she said she was doing the "Tighten Up" to stay loose.
Tyus retired from amateur sports after the 1968 Olympics. In 1973 she was invited to compete in the 60-yard dash in the new Professional International Track Association competitions. In her first-year return, she won eight of eighteen events. The following year, she won every event she entered, a total of twenty-two races. Tyus went on to coach at Beverly Hills High School, and was a founding member of the Women's Sports Foundation.