Although increasing attention has been given to student academic achievement, usually measured by grade point average (GPA), and fitness in children and adolescents, much fewer studies have been conducted in higher education settings, especially in China. This study investigated the longitudinal associations of aerobic fitness (AF), body mass index (BMI), and GPA in Chinese pre-service teachers at a university. A longitudinal research design was employed to track changes in AF, BMI, and GPA, for a total of 1980 students for four years. Multi-level growth models were used to examine the interactive changes of the above three variables. It was found that GPA and BMI increased each year, while AF declined dramatically at the fourth year. The three-way interaction among GPA, gender, and BMI was significant, suggesting females who were overweight/obese had lower GPA. The data from the current study suggested that AF did not impact students’ GPA. Class standing (i.e., the fourth year) and gender (i.e., females) need to be taken into consideration when designing interventions to improve student overall fitness and academic performance in Chinese pre-service teacher populations.