Objective: To assess the changes of students’ hand washing behaviors after implementing comprehensive interventions, and to provide the reference for hand-washing promotion among students. Methods: The implementation of comprehensive intervention projects had been carried out in three aspects: hardware transformation, system establishment, health management and health education. A randomized controlled trial method was used to select each one primary and secondary school in 2016 from 17 districts in Shanghai, and 17 intervention schools and 17 control schools were taken as object. Non-participant observation was used for students’ hand washing behaviors for 4 times during May, 2016 to May, 2017. Descriptive analysis and chi square test were used to analyze the data. Results: Compared with the control group, the hand washing rate of the intervention group was lower than that of the control group at baseline. The intervention group has got a higher rate than that of the control group in the evaluation of 1, the assessment 2 and the assessment 3 after intervention. The difference was statistically significant(χ2=7.70, 24.23, 38.88, 21.71, P<0.05). Compared with the control group, the hand washing rate of the intervention group after class was lower than that of the control group. After the intervention, the rate of the intervention group was higher than that of the control group in the evaluation 1, the assessment 2 and the assessment 3 after the intervention. The difference was statistically significant (χ2=8.52, 33.79, 54.64, 13.99, P<0.05). Compared with the control group, there was no difference between the baseline intervention group and the control group before meals. The intervention group after intervention was higher than the control group in the evaluation of 1, evaluation 2 and evaluation 3. The difference was statistically significant (χ2=45.01, 42.19, 27.82, P<0.05). Conclusion After comprehensive intervention, the effect was significant under different temperature conditions, and the effect of intervention lasted for one year.