Abstract: OBJECTIVE To explore the effect of ATP bioluminescence assay on observation of the relationship between hand hygiene of delivery room staff and postpartum nosocomial infection. METHODS Totally 90 doctors, nurses and interns of delivery room were recruited as the study objects and divided into the control group and the study group by the self-controlled method. The baseline survey was conducted in the control group, the acquisition of knowledge of hand hygiene and the hand hygiene compliance at different time points were monitored and evaluated; the ATP bioluminescence assay was performed in the stud group. The rate of initiative hand washing, qualified rate of hand hygiene and incidence of nosocomial infection were observed and compared between the two groups of health care workers before and after the intervention. RESULTS Before the intervention, there were no significant differences in the rate of initiative hand washing and qualified rate of hand hygiene among the health care workers at the different time points ; the rate of initiative hand washing and qualified rate of hand hygiene were significantly higher in the observation group than in the control group after the intervention (P＜0.05); there was no significant difference in the incidence of nosocomial infection between the observation group and the control group before the intervention; the incidence of nosocomial infection of the observation group was significantly lower than that of the control group after the intervention (P<0.05). CONCLUSION The onsite monitoring and feedback of hand hygiene of the delivery room staff based on the ATP bioluminescence assay may effectively raise the hand hygiene compliance and qualified rate of hand hygiene of the health care workers and reduce the incidence of postpartum nosocomial infection, and it is worthy to be promoted in the hospital.