In college, Tyus continued to win races. Her win at the National AAU women's outdoor meet in the 100-meter race earned her a spot on the 1964 Olympic track team. Her TSU teammate Edith McGuire was also on the Olympic team that year. The games were played in Tokyo, and McGuire was favored to win. Tyus sped past McGuire in the final heat to win the gold medal. She was proud of her accomplishment, but that pride was tempered by the certain knowledge that she was expected to be a woman first. She explained to Votava, "Coach Temple always told us that we could break world records and win gold medals, but we probably still wouldn't be recognized because we were women and we were Black."
Those words became truer in 1968 during the Olympic games held in Mexico City, Mexico. Tyus, who had been encouraged by her family to quit before then, was again competing for the United States track team. She was determined to defend her 100-meter race championship despite suggestions that at twenty-three she was too old to be competing. She was also troubled by her choice to compete as a representative of the United States during a time of extreme racial turmoil. Many black athletes from the U.S. had boycotted the games. Tyus chose to go, but protested silently by always wearing black during awards ceremonies.
At the games, Tyus set the world record with her time in the 100-meter race at 11.08 seconds and secured a record-breaking gold medal in the same race. She also contributed to the 4×100-meter relay team that won the gold medal. Her time in the 100-meter race would not be topped by another American athlete until 1984. In addition, she became the first person to defend the 100-meter race title in successive Olympics. That record would not be broken until Carl Lewis won his second Olympic gold for the race in 1988.
In the climate of protest that existed during the 1968 Olympics, Tyus's accomplishments passed by unnoticed. Wanting to register protest against the expulsion of Tommie Smith and John Carlos, U.S. track runners who had raised their arms in a Black Power salute during an awards ceremony, Tyus gave her medals to Smith and Carlos. Years later she described her admiration to People, "What I did … was win a track event. What they did lasted a lifetime, and life is bigger than sport." Tyus continued competing on the amateur circuit until 1973 when she started competing professionally. In 1974, she set the world indoor track record for the 70-meter race at 8.3 seconds.