Background Depressive moods among occupational population are prevalent, which seriously affect their mental-physical health and socioeconomic productivity. This has become an urgent public health concern. Objective To understand current situation of depressive mood among an occupational population aged 18 to 60 covering 120 cities of China, and to explore the relationship between work-family conflict and depressive mood as well as the role of life stress in the relationship, as to provide a scientific basis for developing measures to reduce depressive mood in the occupational population. Methods Using the data of the Psychology and Behavior Investigation of Chinese Residents in 2021, an occupational population aged 18 to 60 years was selected as study subjects. A total of 3785 subjects were included in the study. The Work-Family Conflict Scale, Subjective Life Stress Scale, and Patient Health Questionnaire-Depression Scale were used to assess the work-family conflict, life stress, and depressive mood of the subjects. Harman's single-factor method was used to evaluate common method bias in the survey data. One-way ANOVA was applied to test the difference in work-family conflict, life stress, and depressive mood among occupational population by demographic characteristics, and chi-square test was used to analyze the difference in reporting depressive mood. Partial correlation analysis was used to evaluate the correlation between selected variables. Process plug-in was used to test potential mediating effect of life stress on work-family conflict and depressive emotion. Results The scores of work-family conflict, life stress, and depressive mood among the occupational population were 24.60±8.28, 9.28±3.78, and 5.43±5.30, respectively. The prevalence rates of depressive mood was 48.7% (1888/3875). The score of depressive mood among occupational population who never smoked was significantly higher than that of those who smoked (P<0.01). The partial correlation analysis showed that work-family conflict and life stress were positively correlated with depressive mood (r=0.472 and 0.447, P<0.001); work-family conflict was positively correlated with life stress (r=0.361, P<0.001). The mediation analysis results showed that work-family conflict positively associated with life stress (b=0.361, P<0.001); life stress positively associated with depressive mood (b=0.318, P<0.001); work-family conflict positively associated with depressive mood (b=0.357, P<0.001), and played a partial mediating role in the relationship between work-family conflict and depressive mood with an effect size of 0.115 (95%CI: 0.099, 0.130) that accounted for 24.36% of the total effect. Conclusion Work-family conflict may not only directly affect depressive mood among occupational population, but also indirectly affect depressive mood through a mediating effect of life stress.