PurposeThis study aims to investigate the association between domain-specific physical activity (PA), sedentary behavior, subjective health perception, and health-related quality of life (HR-QoL) in Korean adults aged ≥ 65 years.MethodsThis cross-sectional study analyzed 6,004 older adults from the Korean National Health and Nutrition Examination Survey 2017–2020. PA and sedentary behavior were measured using a global PA questionnaire, and HR-QoL was assessed using the EuroQol-5 Dimension (EQ-5D, three-level version). Multiple logistic regression was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) after adjusting for confounding parameters.ResultsOlder adults who were physically active at work showed a negative association with subjectively good health and HR-QoL, whereas those physically active in transport or leisure time showed a positive association with subjectively good health and HR-QoL. Older adults highly engaged in sedentary behavior showed a worse perception of health and HR-QoL. Compared to high sedentary behavior and physical activity during leisure time or transport, the EQ-5D index was higher than that of their counterparts.ConclusionBoth domain-specific PA and sedentary behavior were significantly associated with older adults’ perception of health and HR-QoL. Interventions are needed to improve HR-QoL by reducing sedentary behavior and encouraging physical activity in transportation or leisure time among adults aged 65 years and above.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12955-023-02136-8.