Objective: To investigate the association between mental sub-health and physical fitness of Chinese adolescents, to provide a reference for promoting the physical and mental health of Chinese adolescents. Methods: A total of 16 545 adolescents (13-22 years) were selected by random cluster sampling in six major administrative regions in China. The psychological part of the Multidimensional Sub-health Questionnaire of Adolescents (MSQA) and the physical fitness index test were used to assess mental sub-health and physical fitness, respectively. The chi-square test and Logistic regression analysis were used to analyze and compare the relationships between different levels of physical fitness and mental sub-health. Results: The overall detection rate of mental sub-health status among high-grade physical fitness index adolescents was 11.9%, and the detection rate among low-grade physical fitness index youths was 26.0%, and the difference was statistically significant (χ2=970.73，P<0.05). The overall detection rate among high-grade physical fitness index boys was 11.1%, and that of low-grade physical fitness index boys was 24.4%. The difference was statistically significant (χ2=566.12，P<0.05). The overall detection rate of mental sub-health status among high-level physical fitness index girls was 12.8%, and that among low-level physical fitness index girls was 27.6%, and the difference was statistically significant (χ2=412.25，P<0.05). After adjusting for gender, age, BMI, and region in the Logistic regression model, the detection rate of mental sub-health status of adolescents with low-grade physical fitness index was 2.35 times higher than that of high-grade physical fitness index adolescents(95%CI=2.12-2.61). Conclusion Adolescents with poor physical fitness have a higher risk of mental sub-health.Attention should be paid to adolescent physical fitness development to comprehensively improve physical and mental health development.