Background: The life attitude of health care workers can deeply influence the quality of care. Examining the performance of the Short-Form Life Attitude Inventory (SF-LAI), this study analyzes the factorial structure, reliability, and invariance of the revised SF-LAI across genders and professions among the staff of a teaching medical center. Methods: The SF-LAI was developed for university students in Taiwan. From January to February 2019, we administered a cross-sectional survey of life attitudes by distributing the SF-LAI to all staff members of a medical center in Taiwan. The construct validity was evaluated using a confirmatory factor analysis (CFA). Model fit was assessed in terms of the comparative fit index (CFI), Tucker–Lewis index (TFI), standardized root mean square residual (SRMR), and root mean square of error of approximation (RMSEA). Internal consistency was calculated using Cronbach’s alpha and McDonald’s omega. We also performed the CFA invariance analysis for the SF-LAI-R across genders and professions (physician, nurse and other hospital staff). Results: Of 884 (24.62%) responses, 835 were valid. The participants had a mean age of 47.8 years, and 20.12% were male. In a comparison of multiple CFAs, a second-order model with six factors outperformed other models. The goodness of fit indices revealed the CFI was 0.955, TFI was 0.952, RMSEA was 0.071, and SRMR was 0.038. The Cronbach’s alphas, McDonald’s omega coefficients for internal consistency were all greater than 0.8. The first and second-order model had metric and scalar invariance across genders and professions. Conclusions: As health care demands evolve, humanities are becoming more important in medical education. Life attitude of hospital care worker is a crucial indicator of whether one embodies the ideals of a humanistic education. The revised SF-LAI has acceptable structural validity, internal consistency, and invariance across genders and professions among staff members of a teaching medical center.