Objective To understand the current situation of the use and cognition of personal protective equipment (PPE) by healthcare workers in the isolation wards (rooms) during the coronavirus disease 2019 epidemic, and provide data support for the country to further optimize the configuration of PPE and carry out more targeted training and supervision. Methods In March 2020, healthcare workers in isolation wards (rooms) in provinces and cities in the country were investigated by filling in a questionnaire about the use of different levels of PPE. Results A total of 6 859 questionnaires were collected from 29 provinces and cities, containing 5 935 valid questionnaires (86.53%). Among them, there were 448 assisting-Hubei medical team members and 5 487 non-assisting-Hubei healthcare workers. The actual use rate (74.22%) and intended use rate (73.14%) of surgical masks in the cleaning areas were the highest. The actual use rate and intended use rate of working caps in potentially contaminated areas and contaminated areas were higher than 90%. There were various combinations of protective articles, and there were significant differences between actual use and intention (P<0.05). Except for the boot covers (single layer) in the cleaning areas, the working caps, isolation clothing and boot covers (double layer) in the potential pollution areas, and the protective face screens, working caps, three-layer shoe covers and single/double/three-layer boot covers in the pollution areas-splashing operations (P>0.05), the actual utilization rates of protective articles of the assisting-Hubei medical team members were generally higher than those of the non-assisting-Hubei healthcare workers (P<0.05). Conclusions The instructions for the use of PPE in different areas and operations should be further refined, and standardized training should be increased. The phenomenon of non-standard use of protective equipment should be reduced, and a safety barrier should be provided to the healthcare workers.