Objective To evaluate the effect of fluorescent labeling method on improving the contamination of personal protective equipment (PPE) of health care workers (HCWs) during removing of PPE. Methods 69 HCWs were recruited from 8 tertiary hospitals in Chengdu City for training on PPE wearing and taking off, and the effects of PPE removing were assessed by ultraviolet fluorescent labeling method before training, on the day after training, half a month after training and one month after training, the number and sites of residual fluorescent contamination on inner clothing and skin surface after removing PPE were recorded, changes in contamination points and contamination rate were compared. Results Compared with before training, the number of residual fluorescent contamination points of all HCWs after removing PPE on the day after training, half a month after training and one month after training decreased (Z values were 5.306, 5.813 and 6.641 respectively, all P < 0.001).Contamination rates of female, nurse, HCWs with primary-and medium-grade professional title after training decreased significantly compared with before training (all P < 0.05).The number of HCWs'fluorescent contamination points obtained in three assessments gradually decreased over time (all P < 0.05).Work shoes are most likely to be contaminated du-ring PPE removing. Conclusion HCWs are easy to be contaminated in the process of removing PPE, contamination rate can be reduced and contamination situation can be improved after practical training, fluorescent labeling can be used to observe contamination situation in the process of PPE removing.