Objective To compare the effects of different output power and irradiation times of Er:YAG laser irradiation on cell proliferation, clone formation, migration, and transforming growth factor(TGF)-β1 secretion of human periodontal ligament cells. Methods Human periodontal ligament cells were obtained by enzyme digestion, and the cells at the third to the sixth passages were transferred to a tissue culture flask for the experiments. The cells were irradiated by Er: YAG laser at different output powers and same irradiation time(0, 0.45, 0.60, and 0.75 W, at 10 s) as well as at different irradiation times and same power output(0, 10, 30, and 60 s, at 0.60 W). Methyltetrazolium assay, clone formation assay, wound scratch assay, and enzyme-linked immunosorbent assay were performed to observe the effects of Er:YAG laser on the proliferation, clone formation, migration, and TGF-β1 secretion of human periodontal ligament cells. Results The output powers of 0.45 and 0.60 W can promote cell proliferation, clone formation, migration, and TGF-β1 secretion when the irradiation time is set to 10 s. No significant statistical differences were found between these output powers. In different irradiation time groups, we can obtain better cell proliferation, clone formation, migration, and TGF-β1 secretion results when the irradiation time is set to 10 s. However, we obtain the opposite results when the irradiation time is set to 60 s. Conclusion An appropriate amount of Er:YAG laser irradiation can promote the proliferation, clone formation, migration, and TGF-β1 secretion of human periodontal ligament cells.