Objective: To investigate an association between quality-adjusted life years (QALY) and work-related stressors (job strain, effort/reward imbalance, and poor support from supervisor and coworkers), and estimate loss in QALY caused by these stressors. Methods: A cross-sectional study investigated data from a third-wave survey (in December 2017) of a 2-year prospective cohort study of Japanese workers. At baseline (first-wave survey), 5000 participants were recruited from workers who registered with an internet survey company. A total of 2530 participants responded to the second-wave survey 1 year later. Participants were then further recruited to the third-wave survey. An online questionnaire collected information regarding health-related quality of life (measured by EQ-5D-5L), job strain, supervisor and coworker support (Brief Job Stress Questionnaire), effort/reward imbalance (Effort/reward Imbalance Questionnaire), and demographic variables (age, sex, education, occupation, work contract, smoking, and alcohol drinking). Multiple linear regression analysis of the QALY score calculated from responses to EQ-5D-5L was employed on standardized scores of the work-related stressors and adjusted for demographic variables (SPSS version 26). Results: Data of 1986 participants were analyzed. Job strain (unstandardized coefficient, b = − 0.013, p < 0.01) and effort/reward imbalance (b = − 0.011, p < 0.01) and coworker support (b = 0017, p < 0.01) were significantly associated with QALY score in the fully adjusted model. Conclusion: Job strain, effort/reward imbalance, and poor coworker support may be associated with a reduced QALY score among workers. A substantial impairment in QALY associated with the work-related stressors indicates that workplace interventions targeting work-related stressors may be cost-effective.