Objective: To investigate the clinical effect of subcutaneous separation with small needle knife combined with CO2 dot matrix laser in the treatment of acne scars. Method: 62 patients with acne scars treated in our hospital from November 2024 to August 2025 were selected and grouped according to the treatment plan. The control group (n=31) was treated with CO2 fractional laser; The observation group (n=31) received subcutaneous separation with a small needle knife combined with laser treatment. Compare the impact of clinical efficacy, skin condition, patient satisfaction, repair factors, and complications between two groups. Result: The total effective rate of the observation group was higher (93.55% vs 74.18%, P<0.05). After treatment, the observation group had lower scores in skin texture, pigmentation, purple pigment, and pore size (P<0.05); Higher satisfaction (P<0.05); EGF and bFGF levels of fibroblast growth factor were higher (P<0.05). There was no significant difference in the incidence of complications between the two groups (P>0.05). Conclusion: The combination of small needle knife subcutaneous separation and CO2 dot matrix laser treatment for acne scars is effective, can improve skin condition and patient satisfaction, promote skin repair, and has high safety.