Gender makes a difference in health and physical activity (PA). This research aimed to identify the gender difference in the relationship of PA and subjective happiness among Chinese university students. A cross-sectional survey was conducted in Shanghai Jiao Tong University from July 7 to 17 in 2021, using an anonymous online self-report questionnaire. The questionnaire included the Chinese version of the International Physical Activity Questionnaire-Short Form (IPAQ-SF), the Subjective Happiness Scale (SHS), the Depression Anxiety Stress Scale-21(DASS-21). The demographic and health-related factors were also reported. Linear regression was carried out to identify the relationship of PA and subjective happiness. In total, 1,512 students (1,108 males and 404 females) from three comprehensive schools completed the survey. The SHS score was 17.20 ± 3.44, and it was 17.00 ± 3.23 in males, and 17.75 ± 3.93 in females, respectively ( < 0.001). There was no gender difference in vigorous, moderate PA, or walk min/week, while female students had longer sedentary behavior hours/day than males. Male students scored higher in each subscale of DASS-21. After controlling for confounders, vigorous PA min/week (β = 0.002, = 0.002) and sedentary behavior hours/day (β = 0.168, = 0.005) were significantly positively associated with happiness in male students. In contrast, walk min/week (2= 0.002, = 0.005) was significantly positively associated with happiness in female students. This research demonstrated a significant gender difference in the association between PA and happiness. Policymakers and college management should pay more attention to PA programs to promote students' happiness and mental health status.