ObjectiveTo explore the effect of 5M1E quality analysis toolbased hand hygiene(HH) comprehensive intervention measures on pediatric healthcareassociated infection(HAI).MethodsHH status of 29 health care workers(HCWs) in the pediatric department of a hospital was monitored, AprilJune 2015 was baseline survey stage, 5M1E quality analysis tool was adopted to analyze various factors affecting the compliance rate of pediatric HH, intervention measures began to be taken in June 2015, and compared with HH in July 2015March 2016. The occurrence of HAI between July 2014March 2015 and July 2015March 2016 was compared.ResultsHH compliance rate of HCWs increased from 30.86% in AprilJune 2015 to 81.94% in JanuaryMarch 2016; HH correct rate increased from 68.14% to 93.75%; HH compliance rate and correct rate of HCWs gradually increased（χ2=2 608.626, 630.798，respectively, both P＜0.001）. Qualified rate of detection of HCWs’ HH sampling increased from 20.69% in AprilJune 2015 to 89.66% in JanuaryMarch 2016（χ2=31.957，P＜0.001）. Incidence of HAI decreased from 7.74% in July 2014March 2015 to 3.62% in July 2015March 2016（χ2=46.717，P＜0.001）.ConclusionApplication of 5M1E quality analysis tool in the investigation, analysis, and comprehensive intervention in HCWs’ HH in pediatric department has greatly enhanced HCWs’ HH compliance rate, and reduced the incidence of HAI in pediatric patients.