Objective To evaluate the disinfection efficacy of alcohol-based hand disinfectants for medical glove surface in continuous operation, and explore approaches of improving hand hygiene compliance. Methods Between March and July 2015, and between March and July 2016, all the nurses (n=5) in Blood Collection Room of Outpatient Department with many patients and frequent sterile operation needed were included in this study. Nurses wearing gloves for hand hygiene and disinfection between March and July 2016 were regarded as the observation group, and those who used alcohol-based disinfectants for routine hand hygiene without gloves between March and July 2015 were designated into the control group. The hand hygiene compliance was compared between the two groups. From March to July 2016, alcohol-based hand disinfectants were used for disinfection on the glove surface during continuous blood sampling by nurses with gloves on their hands. Fifty samples were collected respectively after the gloves were used for 1, 2, 5, 10, and 15 continuous times for blood sampling. Then, the samples were sent to the bacteriology room for culture. Results All of the 250 samples were qualified, and the bacterial colonies on the surface of gloves were equal to 10 cfu/mL2 or lower. NoStaphylococcus aureus,Escherichiacoli or other pathogens were detected on the glove surface. However, after the gloves were continuously used for 15 times, the gloves of two nurses could not meet the research criteria again because of significant sweating. Conclusions In the process of continuous blood sampling, it is effective to use alcohol-based hand disinfectants to disinfect the surface of medical gloves, which can promote hand hygiene compliance to a certain extent. However, there is a certain limit on the times of usage for medical gloves.