Summary Most studies investigating the association between physical activity and osteoporosis prevention only focused on specific types of physical activity. This study’s evidence regarding the combined effects or interaction of sleep duration and physical activity. The findings emphasize the role of sleep duration and physical activity in association with osteoporosis. Purpose The associations between physical activity, sleep duration, and prevalent osteoporosis in Taiwanese adults were studied in this cross-sectional study. Methods The Taiwan Biobank enrolled a community-based cohort of ~ 120,000 volunteers (as of April 30, 2020) between 30 and 76 years of age with no history of cancer. Amongst, bone mineral density (BMD) measures by dual-energy X-ray absorptiometry (DXA) were available in 22,402 participants. After excluding individuals who had no complete data of BMI ( n  = 23), MET score ( n  = 207), T-score ( n  = 8,826), and sleep duration ( n  = 16), 13,330 subjects were included as the primary cohort. Univariate and multivariable regression analyses were performed to determine the associations between the presence of osteoporosis, physical activity level, sleep duration, and other variables. Results The results showed that after adjustment, subjects with physical activity < 20 METs/week and ≥ 20 METs/week (aOR = 1.017 and 0.767, respectively) were associated with risk of osteoporosis than those with zero MET. The odds of osteoporosis were not significantly lower in subjects who slept for ≥ 8 h/day (aOR = 0.934,p=0.266). In addition, compared to short sleepers with no physical activity, adults with increased physical activity ≥ 20 METs/week and sleep ≥ 8 h/day had a significantly lowest likelihood of osteoporosis (aOR = 0.702). Those with medium physical activity (< 20 METs/week) plus average sleep duration (6.5–8 h/day) did not have significant higher odds of osteoporosis (aOR = 1.129,p=0.151). Conclusion The findings emphasize the joint role of sleep duration and physical activity in association with osteoporosis. Adults with high physical activity plus high sleep hours have the highest BMD and lowest risk of osteoporosis.