ObjectiveTo investigate the application effect of fluorescence labeling on quality assessment of hospital environmental cleanliness, and provide basis for establishing rational and effective assessment strategy. MethodsFrom October 2014 to February 2015, frequently touched object surface in the wards of a tertiary firstclass general hospital were randomly selected and labelled, after cleaning staff completed the cleaning, effectiveness of hospital environmental cleanliness quality was assessed by fluorescence labeling and microbial culture method (golden standard). Results216 frequently touched sites in the wards were detected, the qualified rates detected by fluorescence labeling method and microbial culture method were 43.06% and 49.54% respectively, the sensitivity and specificity of fluorescence labeling for assessing environmental cleanliness quality were 82.24% and 94.50% respectively, kappa value was 0.88, positive and negative predictive values were 93.61% and 84.43% respectively. Cost of fluorescence labeling method for each sampling point detection was ￥0.15, while microbial culture method was ￥8.20. ConclusionFluorescence labeling can be applied in assessing the quality of hospital environmental cleanliness, it is reliable, economical, and practicable.