Objective: To investigate differences in infection control awareness, and related implementation of measures between anti-epidemic medical staff and workers during the outbreak of novel coronavirus (COVID-19) infection, and to propose corresponding countermeasures. Methods: A self-designed questionnaire on the practice of infection control and implement were conducted, and 250 medical staff and workers participating in the anti-epidemic work in our hospital from March to April 2022 were investigated online. Results: A total of 208 valid questionnaires were recovered, of which 192 were valid questionnaires, including 158 medical staff (82.3%) and 34 (17.7%) workers. Before entering the isolation point, the proportion of workers vs medical staff who knew the knowledge about COVID-19, hospital infection control procedures, standardized use of masks, awareness of occupational post-exposure disposal procedures, awareness of sensory control and implementation of sensory control in living areas were 58.8% vs 89.2%, 67.6% vs 91.1%, 82.4% vs 98.7%, (58.8% vs 98.7%), (47.1% vs 98.7%), (47.1% vs 94.3%) (P<0.05). However, the proportion of discomfort such as sweating, poor breathing, dizziness and fatigue during work in the polluted area was significantly lower than those of medical staff（2.9% vs 11.5%, P<0.001）. Then targeted measures such as scenario simulation training assessment and real-time "one-to-one" on-site supervision were performed,and the infection control practice norms of these two groups have been significantly improved. The proportion of medical staff with knowledge about new coronary pneumonia and aware of the hospital infection system process increased to 99.4% (P<0.001) and to 98.7% (P=0.002), respectively, while of workers increased to 85.3% (P=0.015) and to 100% (P<0.001). The proportion of workers got a score of 90-95 on hospital infection theory and operation increased from 29.4% to 38.2%, and the proportion with a score of 95 or above increased from 32.4% to 58.8% (P<0.001). Conclusions: The implement and and awareness of infection control practice in workers is worse than those of medical staff, and workers are a high-risk group for hospital infection. The scenario simulation training and real-time assessment methods, whole process "one-to-one" on-site supervision can effectively improve the awareness and practical ability of medical staff, especially workers.