Objective To investigate the psychological stress of health care workers(HCWs) in isolation wards during the pandemic period of coronavirus disease 2019 (COVID-19), and provide scientific basis for targeted psychological intervention at the later stage. Methods Psychological stress of HCWs in the isolation wards of a tertiary first-class general hospital in Sichuan Province was surveyed by using Questionnaire Star, questionnaire include basic information and psychological stress-related scale. Results Comparison in score of anxiety factors for infection:nurses were more anxious than doctors(t=2.471,P=0.007). Comparison in fatigue factor score:HCWs who having given birth was higher than not having given birth(t=2.474,P=0.016); nurses were higher than doctors(t=2.714,P=0.009); non-infection specialty was higher than infection specialty(t=-2.513,P=0.015). Comparison in work load factor score:working years ≥ 20 years was higher than<20 years (t=-5.236, P<0.001). Comparison in work motivation factor score:working years ≥ 20 years was higher than<20 years(t=-2.039, P=0.046); work motivation factor score of HCWs who obtained knowledge about COVID-19 through different number of ways was significantly different(F=4.673,P=0.013). Comparison in support system factor score:infection specialty was higher than non-infection specialty (t=2.371,P=0.021); protection according to protective level was higher than protection not by protective level (t=2.153,P=0.035). Overall psychological stress score of HCWs who obtained knowledge about COVID-19 through different number of ways was sifnificantly different(F=6.668, P<0.001). Conclusion In order to make frontline HCWs in isolation wards (especially nurses, HCWs who have given birth, and HCWs of non-infection specialty) to cope with the pandemic at minimum stress in a favorable working environment, knowledge on COVID-19 should be updated and provided timely, psychological support should be given.