Objective To explore the application effect of the "one before and five after" hand hygiene (HH) indicators in HH management of cleaning staff in medical institutions. Methods From December 2021 to January 2022, cleaning staff from a tertiary hospital were selected as the research subjects through convenience sampling method and randomly divided into three groups (control group, experimental group Ⅰ, and experimental group Ⅱ). The control group received training according to the World Health Organization (WHO) "two before and three after" HH indicators, while the experimental group Ⅰ received extensive training according to the "one before and five after" HH indicators, and the experimental group Ⅱ received precision training according to the "one before and five after" HH indicators. HH compliance rate, HH compliance rates for different indicators, and the distribution trend of HH moments among three groups of cleaning staff were compared. Results A total of 26 cleaning staff in the control group, 24 in the experimental group Ⅰ, and 18 in the experimental group Ⅱ were surveyed. HH compliance rates before training in the control group, experimental group Ⅰ, and experimental group Ⅱ were 31.30%, 27.18%, and 30.30%, respectively; and those after training were 43.11%, 59.26%, and 83.62%, respectively. After training, HH compliance rate of cleaning staff in experimental group Ⅱ significantly increased compared to before training, with a growth rate of 53.32% and statistically significant difference (P < 0.05). After training, HH compliance rate in experimental group Ⅱ was the highest (83.62%). The HH moment with the highest HH compliance rate of cleaning staff in experimental group Ⅰ was after unloading personal protective equipment (64.77%), and the lowest was before cleaning and disinfection (54.55%). Except after preparing tools, HH compliance rates of experimental group Ⅱ at other different HH moments after training were all higher than experimental group Ⅰ, differences were statistically significant (all P < 0.05). After training, the HH moment with the highest HH compliance rate of cleaning staff in experimental group Ⅱ was after the disposal of medical waste (90.72%), and the lowest was after the preparation of tools (78.33%). High frequency periods for HH behavior of cleaning staff were 6:00 a.m.-9:00 a.m and 3:00 p.m.-4:00 p.m. Conclusion The "one before and five after" HH indicator is more suitable for monitoring and training on HH compliance of cleaning staff in medical institutions than the "two before and three after" HH indicators. "one before and five after" HH precision training can effectively improve the HH compliance of cleaning staff in medical institutions.