Objective To explore the effect of "3-step rubbing method" adopted by health care workers (HCWs) during the process of removing protective equipment in COVID-19 isolation ward. Methods 35 front-line clinical HCWs who supported the isolation ward of a tertiary first-class hospital in Shanghai from April to June 2022 were selected. Before removing protective equipment, each HCW conducted hygienic hand disinfection with the "3-step rubbing method" and the "6-step rubbing method" once, respectively. SARS-CoV-2 nucleic acid test results of the front, back and finger side of the outermost gloves before and after conducting two hygienic hand disinfection me-thods were compared. Results SARS-CoV-2 nucleic acid test results of the surface of outermost glove before and after disinfected by two hygienic hand disinfection methods were statistically different (both P < 0.05). Positive rate of SARS-CoV-2 nucleic acid test of the surface of the outermost gloves after "6-step rubbing method" and "3-step rubbing method" hygienic hand disinfection were 4.76% (5/105) and 5.71% (6/105), respectively, with no statistically significant difference between the two groups (P>0.05). Standard rate of hand hygiene was higher when using the "3-step rubbing method" than the "6-step rubbing method" (91.43% vs 77.14%, P=0.02). Conclusion The "3-step rubbing method" can achieve the same disinfection effect as the "6-step rubbing method" during the process of HCWs' removing protective equipment in COVID-19 isolation ward, but the standard rate of "3-step rubbing method" for hand hygiene of HCWs is higher, therefore, the "3-step rubbing method" is helpful for ensuring the occupational safety of HCWs, and is easy to be promoted and accepted in clinical practice.