Background: Evidence-based 24-hour movement guidelines have been developed to integrate recommendations for time spent on moderate to vigorous physical activity (MVPA), screen time (ST), and sleep. This study investigated the adherence to 24-hour movement guidelines and explored the relationship with health-related quality of life (HRQoL) in adolescents. Methods: Cross-sectional findings are based on 1774 adolescents (49.1% female) aged 13–16 years from the Shenyang region of China. MVPA, ST, and sleep duration were self-reported using the International Physical Activity Questionnaire, an ST questionnaire, and a wake–sleep time questionnaire. HRQoL was measured using the Pediatric Quality of Life Inventory, which encompasses physical, psychosocial, and total HRQoL. Adherence to 24-hour movement guidelines was classified by meeting general (eg, none, 1, 2, and 3 guidelines) and specific combinations (eg, MVPA and ST guidelines) within the 3 recommendations. Associations were examined using general linear models adjusted for age, sex, and parents’ education. Results: A total of 5.1% of the sample met all three 24-hour movement guidelines. Additionally, compared with meeting no guideline, meeting all 3 guidelines was associated with better physical (β = 5.37; 95% CI, 0.69–9.49; P = .005), psychosocial (β = 3.76; 95% CI, 0.84–6.68; P = .012), and total HRQoL (β = 4.16; 95% CI, 1.35–6.98; P = .004). Compared with not meeting the specific combination guidelines, meeting MVPA and ST was associated with better physical HRQoL (β = 5.52; 95% CI, 1.30–9.74; P = .01). Conclusions: These cross-sectional findings suggest that meeting all three 24-hour movement guidelines were associated with better HRQoL in adolescents. Since a low proportion of this sample was meeting the overall guidelines, future effort should encourage, and perhaps improve adherence to the 24-hour movement guidelines.