To explore the effect of CO2 fractional laser combined with tacrolimus in the treatment of vitiligo. Methods A total of 70 patients with vitiligo admitted to our hospital from January 2020 to December 2021 were selected as the research objects. According to the random number table method, they were divided into control group and observation group, with 35 patients in each group. The control group was treated with compound glycyrrhizin tablets combined with tacrolimus, and the observation group was treated with CO2 fractional laser combined with tacrolimus. The oxidative stress indexes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA)], Treg cells, related cytokine forkhead box protein P3 (Foxp3), clinical effect and complications were compared between the two groups. Results After treatment, SOD, GSH-Px, MDA, Treg cells and Foxp3 in the observation group were better than those in the control group (P <0.05). The total effective rate of treatment in the observation group was 97.14%, which was higher than 82.86% in the control group (P <0.05). The incidence of complications in the observation group was 17.14%, which was lower than 42.86% in the control group (P <0.05). Conclusion CO2 fractional laser combined with tacrolimus is effective in the treatment of vitiligo, which can effectively reduce stress injury, improve immune function, and has high safety.