Objective Toxoplasma gondii is a worldwide protozoan parasite that could infect virtually all warm-blooded animals, including humans. Our study aimed to investigate the prevalence of T. gondii infection in college students at Anhui province, China and to assess risk factors for T. gondii infection in college students. Moreover, growing studies demonstrated the association between T. gondii infection and host behavioral changes. We also studied the linkage between T. gondii and physical education (PE) scores of college students. Methods A total of 2704 serum samples of medical school students attending physical education lessons were collected from September 2017 to September 2019 and evaluated for T. gondii IgG antibodies using an enzyme-linked immunosorbent assay (ELISA). Questionnaires and statistical analysis were used to determine the risk factors for T. gondii infection. We also analysed PE scores of T. gondii -infected students and T. gondii-uninfected students. Results The overall seroprevalence of T. gondii was 11.5%. The main risk factors related to T. gondii infection in college students were cat in the household and gardening or agriculture activity. Furthermore, in the basketball group and the soccer group, scores of T. gondii seropositive students were significantly higher than those of seronegative students, while in other sports there was no difference between scores of T. gondii-infected students and T. gondii uninfected students. Conclusion This is the first report of T. gondii seroprevalence in college students in Anhui province, China.