This study aimed to investigate the difference between the self-perceived and measured physical fitness of university students majoring in science and engineering. A total of 353 healthy university students (252 men and 101 women) were studied. Health-related physical fitness variables, such as body composition, cardiovascular endurance, muscular strength, muscular endurance, and flexibility, were assessed using the Self-Appraisal Questionnaire and actual tests including bioelectrical impedance analysis, ergoracer bicycle, handgrip strength, sit-up, and sit-and-reach test. The students’ self-perceived and measured physical fitness were compared, and the k value of the dichotomous variables was calculated to evaluate the agreement of the two measurement methods. There were differences between self-perceived and measured physical fitness in male and female university students, as shown by their Body mass index, ergoracer bicycle, handgrip strength, sit-up, and sit-and-reach tests. In addition, the agreement was low in both males (k=0.21) and females (k=0.22). These results suggest that since their perceived physical fitness could give students an incorrect understanding of their fitness level, it is necessary to inform them of their actual fitness level based on measured physical fitness, and motivate them to increase the frequency of physical activity and exercise in their daily lives.