(1) Background: The COVID-19 epidemic had caused more than 100 million confirmed cases worldwide by the end of January 2021. The focus of this study was to explore which stress was felt the most by nursing staff in isolation wards in the face of dangerous infectious diseases. (2) Methods: Nursing staff in negative pressure isolation wards were taken as the research objects. The sources of stress were divided into 14 items in three categories, namely, patient care, infection protection, and

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... rt system, and the questionnaire results were ranked by a Gaussian curve. (3) Results: Even during the COVID-19 epidemic, nurses in isolation wards still consider that the clinical symptoms of patients in isolation wards cannot be closely tracked as the primary consideration. (4) Conclusions: During the epidemic period, the ability and confidence of nursing staff were strengthened through education and training, and their chances of infection were reduced through comprehensive vaccination and the improvement of protective equipment. In the face of the unstable mood of patients and their families due to isolation, more protective measures should be prepared for nursing staff. In order to relieve the stress, supervisors can adjust the nursing manpower timely according to the difficulty and risk of patient care to reduce the care stress.