Objective To investigate the knowledge, attitude and behavior(KAB)of hand hygiene(HH) among caregivers in a tertiary first-class children's hospital, and analyze the changing trend of KAB and its risk factors. Methods From January 2016 to December 2018, the implementation of HH among caregivers in a children's hospital was investigated by concealed on-site observation, bacteriological surveillance on HH was conducted, KAB of HH and its influencing factors were performed questionnaire survey. Results Caregivers' HH compliance rate was 71.10%, qualified rate of bacteriological surveillance on HH was 72.92%, there were no significant differences in the compliance rate and qualified rate of HH among caregivers in three years (all P>0.05). In 2016, caregivers' HH knowledge score and attitude score was correlated (P<0.05), but knowledge score and attitude score were not correlated to behavior score (both P>0.05); in 2017-2018, caregivers' HH knowledge score and attitude score were both correlated to behavior score (both P<0.05). Insufficient supervision of hospital, considering the irritation of skin by alcohol-based hand rub and caregivers' weak consciousness of hand-washing were main factors for lower scores of HH behavior. Conclusion The overall HH compliance rate in this children's hospital is relatively high, but there is still room for improvement, competent department should formulate feasible HH system and standards, and carry out the supervision on implementation, further improve HH compliance rate of caregivers and qualified rate of bacteriological surveillance.