Existing studies have separately explored the association between specific intensity and domain of physical activity (PA) and biological age (BA), potentially impeding more precise guide. We used data of 5216 middle-aged and older adults aged 45 and over to explore and compare the relationship of different domains, intensities, and doses of PA and BA. Transport-related walking and leisure-time moderate-intensity PA (MPA) were significantly associated with younger BA, rather than other intensity of work-related PA. There were U-shaped dose-response relationships between transport-related BA or leisure-time MPA and BA. In subgroup analyses for older adults, all intensities of PA were significantly associated with decreased BA. These findings highlighted the importance of considering the domain, intensity, and dose of PA in designing effective age-delaying interventions.