Purpose To investigate the association between preschool children's 24-h movement behaviors and health-related quality of life (HRQoL), exploring the impact of time reallocation among these behaviors and examining gender differences. Methods This study analysed HRQoL and 24-h movement behaviors of 349 preschool children in three kindergartens in Beijing, China, selected through convenience sampling using a cross-sectional study design. A t-test and multivariate analysis of variance methods were used to investigate gender differences. The study examined the relationship between 24-h movement behaviors and HRQoL using component data analysis and component isochronic substitution model methods, with an investigation into gender differences in the overall association. Results The study found a negative correlation between sedentary behavior (SB) and overall HRQoL score (gamma = - 11.92, p < 0.05) in the entire sample, particularly affecting physical health score (gamma = - 14.39, p < 0.01). Among boys, SB was negatively correlated with the HRQoL total score (gamma = - 15.83, p < 0.05), while sleep was positively correlated with psychosocial health scores (gamma = 17.814, p = 0.01). However, there was no significant association found between 24-h movement behaviors and HRQoL in girls. When using the component isochronic substitution model, reallocating 30 min from sedentary behavior to sleep increased the total HRQoL score of preschool children by 0.865 points (95% CI 0.071, 1.658). In contrast, reallocating 30 min from sleep to sedentary behavior resulted in a decrease of 0.850 points (95% CI - 1.638, - 0.062) in the total HRQoL score. Conclusions To improve preschool children's HRQoL, it is recommended to reduce their sedentary behavior time and increase their sleep time. Public health policymakers should consider this when developing 24-h movement behavior guidelines for preschoolers.