Objective To investigate the clinical effect of CO2 fractional laser combined with pimecrolimus cream in the treatment of vitiligo. Methods A total of 80 patients with vitiligo admitted to Qinghai Red Cross Hospital from January 2020 to June 2023 were randomly divided into observation group and control group, with 40 patients in each group. The control group was treated with pimecrolimus cream for external use, and the observation group was treated with CO2 fractional laser on the basis of the control group. The T lymphocyte level, white spot area, quality of life (DLQI) score, oxidative stress index and safety were compared between the two groups. Results After treatment, CD3+, CD4+, CD8+ and CD4+/CD8+ in the two groups were better than those before treatment, and those in the observation group were better than those in the control group (P <0.05). After treatment, the white spot area and DLQI of the two groups were lower than those before treatment, and those in the observation group were lower than those in the control group (P <0.05). After treatment, SOD, MDA and GSH-Px in the two groups were better than those before treatment, and those in the observation group were better than those in the control group (P <0.05). The incidence of adverse reactions in the observation group was 12.50%, which was slightly higher than 5.00% in the control group, but the difference was not statistically significant (P >0.05). Conclusion The effect of CO2 fractional laser combined with pimecrolimus cream can regulate the level of T lymphocytes for patients with vitiligo, reduce the area of leukoplakia, improve the quality of life of patients, reduce the level of oxidative stress, and has high safety, which is worthy of clinical application.