The Depression Anxiety Stress Scale-21 is an instrument in the assessment of mental health status. The current study recruited 1,532 Chinese hospital workers [74.4% female; mean age = 31.97 years] to examine the reliability, latent structure, and measurement invariance of the DASS-21 between genders. The Cronbach’s α values were greater than 0.90 for total score. This study examined four possible models of the DASS-21 using the confirmatory factor analysis in Chinese hospital workers. The results from CFA revealed that the latent (...) structure of the DASS-21 in medical staffs is best represented by a one-factor model. Then we used the one-factor model to examine measurement invariance across genders by using a multiple-group categorical CFA. All values of root mean square error approximation were less than 0.08, all Comparative Fix Index and Tucker–Lewis Index values were greater than 0.90, all ΔCFI values were less than 0.010, and ΔRMSEA were less than 0.015. These findings supported the gender invariance of the DASS-21 among Chinese hospital workers. (shrink)