This study investigated the dose–response relationship between structured physical activity frequency and improvements in physical fitness in preschool children. Sixty-three children (3-6 years) were divided according to weekly participation frequency in a 12-week structured physical training program (60 min/session): low frequency (L, ≤1 session/week), moderate frequency (M, 1-2 sessions/week), moderate-to-high frequency (MH, 2-3 sessions/week), and high frequency (H, ≥3 sessions/week). Physical fitness was assessed before and after the intervention using six standardized motor tests. These tests included walking the balance beam, 10 m × 2 shuttle run, tennis throwing, continuous jumping with both feet, standing long jump, and sit-and-reach. A 2 (Time: pre- vs post-intervention) × 4 (Group: L/M/MH/H) repeated-measures ANOVA was used to analyze changes within and between groups.Significant time × group interactions were found for walking the balance beam, 10 m × 2 shuttle run, tennis throwing, continuous jumping with both feet, and standing long jump (p < 0.05), but not for sit-and-reach flexibility. Post-hoc analyses revealed that the H group performed better than L across all five indicators and outperformed M in the 10 m × 2 shuttle run, tennis throwing, and standing long jump (p < 0.05). The MH group demonstrated better performance than the L group in the balance beam walk, 10 m × 2 shuttle run, and tennis throwing. However, no significant differences were observed between the MH and H groups at post-test. These findings indicate that participating in structured physical activity fewer than two sessions per week is insufficient to enhance preschool children’s physical fitness. In contrast, engagement at least twice per week significantly improves key indicators of strength, coordination, and agility. However, performance reach a performance plateau when frequency exceeds three sessions per week, suggesting diminishing marginal returns. Incorporating moderate-to-high frequency structured physical activity into preschool curricula may thus be an efficient strategy for optimizing fitness development in early childhood. Taken together, these findings emphasize the need for at least two weekly sessions of structured physical activity to effectively enhance physical fitness in preschool children.