Background: Although Perceived Stress Scale (PSS, Cohen, Kamarack, Mermelstein, 1983) has been validated and widely used in many domains, there is still no validation in sport by comparing athletes and non-athletes and examination of related psychometric indices. Purpose: The purpose of this study was to examine the measurement invariance of PSS between athletes and non-athletes, and examine construct validity in the sport contexts. Methods: Study 1 sampled 359 college student-athletes (males = 233; females = 126) and 242 non-athletes (males=124; females=118) and examined factorial structure, measurement invariance and internal consistency. Study 2 sampled 196 student-athletes (males = 139, females = 57, Mage =19.88 yrs, SD = 1.35) and examined discriminant validity and convergent validity of PSS. Results: Results found that 2-factor PSS-10 fitted the model the best and had appropriate reliability. Also, there was a measurement invariance between athletes and non-athletes; and PSS positively correlated with athlete burnout and life stress but negatively correlated with coping efficacy provided evidences of discriminant validity and convergent validity. Discussion: It is suggested that 2-factor PSS-10 can be a useful tool in assessing perceived stress either in sport or non-sport settings. We suggest future study may use 2-factor PSS-10 in examining the effects of stress on athletic injury, burnout, and psychiatry disorders.