Objective This present study aimed to investigate the relationship between working hours and anxiety/depression mood of medical staff in China during COVID-19 epidemic. Methods The cross-sectional interview study was conducted during the period between February 14th and February 29th, 2020. A total of 291 Chinese medical professionals were recruited from 4 cities and participated in the study. Results In 291 participants, 116 (40.0%) medical staff experienced anxiety and 151 (51.8%) underwent depressed mood. In male, the level of GAD-7 and PHQ-9 scores increased with the elevation of working hours per day (WHPD) (β=0.579, p=0.003 and β=0.943; p=0.001) respectively. In female, nonlinear relationship mode was demonstrated. The levels of GAD-7 and PHQ-9 scores increased with the elevation of working hours when it was above 5 hours (β=1.432; p<0.001 and β=1.177; p<0.001), but it did not have a significant association with WHPD when it was less than 5 (p>0.05). Conclusion During the COVID-19 epidemic, we found a strong correlation between the psychological mood and WHPD. The correlation followed different modes in male and female medical workers. Enforcing an upper time limit of WHPD may help decrease the risk of pandemic-related psychological problems in medical workers.