Photobiomodulation (PBM), which stimulates cellular functions through light emission, has recently attracted significant attention in regenerative medicine. This study explores the effects of PBM with two wavelengths on human adipose-derived stem cells (ADSCs) and the characteristics of their secreted exosomes in vitro. ADSCs were isolated from human adipose tissue and treated with PBM with 650 nm and 810 nm wavelengths. The therapeutic effects, including cell viability, proliferation, and exosome characteristics, were evaluated using MTT assays, scanning electron microscopy (SEM), BCA assays to determine exosomal protein concentration, and Dynamic Light Scattering (DLS) to assess exosome size distribution. The results indicated that PBM at both wavelengths significantly enhanced ADSCs viability and proliferation, as confirmed by the MTT assay (p = 0.008 for 650 nm laser and p = 0.007 for 810 nm laser). Additionally, exosome analysis revealed that exosomes from PBM-treated groups exhibited a higher protein concentration and an average size of around 86 nm, as measured by BCA and DLS. We concluded that PBM with 650 nm and 810 nm wavelengths individually significantly enhances the proliferation and survival of ADSCs and improves exosome quality in vitro. The treatment increased exosomal protein concentration and improved size distribution, reflecting enhanced cell function. These findings suggest that PBM could be a promising stimulatory approach for improving ADSC-based therapies in regenerative medicine, particularly for tissue repair and chronic wound healing. Further research is needed to explore the molecular mechanisms and clinical applications of ADSCs under the influence of PBM.