This study aimed to analyze the effects of physical activity (PA), sleep quality, and sedentary behavior on subthreshold depression (StD) among undergraduates. This study included 834 undergraduates and assessed the impact of PA time, sleep quality, and sedentary behavior on depression. The receiver operating characteristic (ROC) analysis was performed to determine cut-off values for StD risk, while the isochronous substitution analysis was performed to evaluate the effects of different activities on depression regulation. Gender, age, and academic grade had no significant influence on depression levels among undergraduates (p > 0.05). However, students engaging in sedentary behavior for more than 12.1 h per day or with a Pittsburgh Sleep Quality Index score above 3.5 were at an increased risk of subclinical depression. Additionally, the isochronous substitution of light-intensity physical activity for other activities (sleep, sedentary behavior, moderate and vigorous intensity physical activity) showed statistically significant effects (p < 0.05) in both 5-min and 10-min substitution models, demonstrating a positive effect on alleviating depression. The findings indicate that specific lifestyle factors, particularly high levels of sedentary behavior and poor sleep quality, are crucial determinants of subclinical depression among undergraduates, independent of demographic variables such as gender, age, and academic grade. Notably, light-intensity PA plays a key role in StD regulation, as substituting it with more intense physical activities or improving sleep quality substantially reduces depression scores. Furthermore, the benefits such substitution became more pronounced with the increase in duration of the activity.