Objective: To explore the relationship between isotemporal substitution of 10 min/d of vigorous physical activity (VPA), moderate physical activity (MPA), walking, sedentary behavior (SB) and sleep (SLP) and depression among vocational school students with different duration of moderate-to-vigorous physical activity (MVPA), so as to provide time allocation suggestions for reducing depression levels. Methods: The convenient cluster sampling method was adopted to conduct a questionnaire survey among 8 149 grade one to grade three students in 14 vocational schools in Shanghai and Jiangsu Province from December 2021 to January 2022. According to whether the MVPA reached 60 min/d, the vocational school students were divided into the MVPA standard group and the MVPA non-standard group, and the isotemporal substitution model was used for analysis. Results: About 19.81% of students were in the MVPA standard group. In the MVPA non-standard group, substituting MPA for all other studied behaviors and substituting SLP for walking and SB were negatively correlated with depression ( β =-0.78, -0.90, -0.88, -0.83; -0.07 , -0.05, P <0.05), and the association of MPA substitution was much greater than that of SLP substitution. In the MVPA standard group, replacing VPA, walking and SB with SLP were all negatively associated with depression ( β =-0.23, -0.12, -0.10 ), whereas replacing MPA, SB and SLP with VPA was all positively associated with depression ( β =0.15, 0.13, 0.23) ( P <0.05). Conclusion The MVPA level of vocational school students is low. The effects of isotemporal substitution for VPA and MPA are different when MVPA duration is up to standard and when MVPA duration is not up to standard. Appropriate time allocation suggestions should be provided based on the characteristics of adolescents with different MVPA durations.