Objective To analyze the effect of plan-do-check-action (PDCA) management mode on the management of healthcare-asssociated infection(HAI) in neonatal intensive care unit (NICU). Methods Children who were admitted to NICU in a hospital were selected as the research objects, those who were admitted between January and June 2018 were in control group, from July to December 2018 were in trial group. Trial group adopted PDCA cycle program to improve the quality of HAI management, HAI in two groups of neonates before and after the implementation of PDCA was compared, hand hygiene of health care workers(HCWs) before and after implementing PDCA was monitored. Results There were 1 254 neonates in control group, 228 were premature neonates, 1 368 neonates in trial group, 304 were premature neonates. Case incidence of HAI in neonates in NICU after implementing PDCA was lower than before implementation (4.09% vs 6.30%, χ²=6.521,P<0.05). Case incidence of HAI in premature neonates after implementing PDCA was lower than before implementation (4.28% vs 8.77%, χ²=4.526,P<0.05). After the implementation of PDCA, the qualified rates of hand hygiene of doctors and nurses were both higher than those before the implementation, difference was statistically significant (P<0.05). Before implementing PDCA, daily consumption of hand disinfectant and hand sanitizer in NICU were 16.94 mL/bed-day and 15.41 mL/bed-day respectively; after implementing PDCA, daily consumption were 25.95 mL/bed-day and 17.81 mL/bed-day respectively. Conclusion Application of PDCA management can improve the quality of HAI management in NICU, and reduce the incidence of HAI, which is worthy of clinical application.