Objective To investigate the role of scar epithelial cells and its potential molecular mechanisms in the efficacy of low-energy CO₂ fractional laser treating post-burn scars. Methods The model of post-major burn scars on the back of rat was established. Three rats with post-major burn scars received 30 mJ low-energy CO₂ fractional laser treatment to detect the activation of scar epidermal cells. Epidermal tissue of scars was isolated for RNA sequencing to screen activated pathways. Subsequently, 18 rats with post-major burn scars were randomly divided into 3 groups (n＝6): the control group without laser treatment, the laser group receiving 30 mJ CO₂ fractional laser treatment, and the laser＋inhibitor group receiving laser treatment and intra-scar injection of IWR-1 (a Wnt/β-catenin pathway inhibitor), to verify the activation status and effects of the selected pathways. Hematoxylin-eosin staining, Masson staining, and Western blotting were used to detect the proliferation of epithelial cells and fibroblasts, the activation of Wnt/β-catenin pathway, as well as the improvement of scar profiles. Results After low-energy laser treatment, there was a significant increase in the number of Ki67-positive, proliferating cell nuclear antigen (PCNA)-positive, cytokeratin 19 (CK19)-positive, and p63-positive cells in the scar epithelial tissue. RNA sequencing coupled with literature analysis identified Wnt/β-catenin pathway as a potential candidate pathway. In the confirmatory experiment, compared to the control group, the Wnt/β-catenin pathway was activated in scar epithelial cells in the laser group 5 d post-laser intervention. After 30 d laser intervention, dermal collagen exhibited a more loosened arrangement, with reduced dermal thickness and significantly less α-smooth muscle actin (α-SMA)-positive fibroblasts compared to the control group. CollagenⅠ, collagen Ⅲ, and the relative ratio of collagen Ⅰ to Ⅲ in the laser group were at a lower level than those in the control group. Administration of the Wnt/β-catenin pathway inhibitor blocked the activation of the Wnt/β-catenin pathway induced by low-energy laser, the proliferation of scar epithelial cells and the improvement of scar profiles. Conclusion Low-energy CO₂ fractional laser treatment can activate the Wnt/β-catenin pathway of scar epithelial cells, thereby activating epithelial cells and yielding significant scar improvements.