Stem cells derived from human orbicularis oculi muscle (hOOM) are a valuable resource for cell therapy. However, when stem cells are continuously cultured, their abilities tend to deteriorate over time. One method to address this issue is to use basic fibroblast growth factor (bFGF) to maintain the stem cell functionality. The limitation is that bFGF is unstable under mammalian cell culture conditions with a half-life of only 8 hours, which poses a significant challenge to the production and maintenance of high-quality stem cells. In this study, we used thermostable bFGF (TS-bFGF) and demonstrated that hOOM-derived stem cells cultured with TS-bFGF exhibited superior proliferation, stem cell function, reduced reactive oxygen species, and cellular senescence delay effect compared to cells cultured with wild-type bFGF. Considering the pivotal role of stem cells in broad ranges of applications such as regenerative medicine and cultured meat, we anticipate that TS-bFGF, owing to its thermostability and long-lasting properties, will contribute significantly to the acquisition of high-quality stem cells.