Objective To understand the current situation of protective mask wearing behavior of health care workers (HCWs), analyze the influencing factors for the failure to wear medical protective masks in a standard manner, and provide basis for the improvement of mask-wearing related training. Methods From June 2022 to March 2023, staff in a tertiary first-class hospital were selected as the research object. Real-time quantitative fitness testing using aerosol condensation particle counting method was applied to test 5 commonly used medical protective masks available in the market. Fitness factor changes of the testing instrument and assistance from professional personnel were needed to comprehensively estimate the wearing condition of medical protective masks. Participants were surveyed through a self-made general information questionnaire. Heads and faces of participants were scanned by three-dimensional (3D) laser scanning technology, and scanned images were imported into Geomagic Studio 2013 software to measure head and face dimensions. Results A total of 222 HCWs were investigated, 991 real-time tests and 208 times of 3D scanning were conducted. 221 (22.30 %) tests showed failure of participants in wearing masks in a standard manner. The non-standard wearing rates of 5 types of medical protective masks were 30.56%, 25.62%, 25.87%, 23.15%, and 7.35%, respectively. The non-standard mask-wearing rates showed statistically significant difference between groups categorized in terms of medical protective masks with different shapes, participants' occupation, time of last training for wearing medical protective masks, and participants' experience in prevention and control of respiratory infectious disease (all P < 0.05). There were no statistically significant differences in non-standard mask-wearing rate between groups with different brands and sizes of medical protective masks, as well as gender and department of participants, etc. (all P>0.05). The body mass index (BMI) was significantly different among participants who wear foldable medical protective masks in the standard and non-standard manner (both P < 0.05). Conclusion Wearing medical protective masks by HCWs in a non-standard manner is influenced by multiple factors. It is recommended to conduct real-time testing before formal quantitative fitness testing, so as to save time and improve testing efficiency. When conducting training on wearing medical protective masks in the future, targeted training should be provided based on mask shape and focus on logistics personnel, interns, individuals with high BMI, those who have never received training on wearing medical protective masks, and those who have never participated in the prevention and treatment of respiratory infectious diseases.