The invention discloses a skeletal muscle stem cell serum-free medium and a preparation method and application thereof. The skeletal muscle stem cell serum-free medium, with the pH value ranging from 7.4 to 7.4, comprises DMEM(dulbecco's modified eagle medium)/F12, serum replacement, cytokines, vitamins, amino acid, recombinant human insulin, mepiquat chloride and 6-chondroitin sulfate. The invention further discloses the preparation method of the skeletal muscle stem cell serum-free medium. Specifically, the serum replacement refers to animal-free component which is defined, thus, serum caused pathogenic risk and instability of different batches and the like are avoided. The skeletal muscle stem cell serum-free medium is applicable to human skeletal muscle stem cell primary culture and scaled amplification culture and has the advantages of high purity, quick cell proliferation, high expansion efficiency, capability of realizing steady passage of at least 20 generations and the like. In addition, the skeletal muscle stem cell serum-free medium effectively improves skeletal muscle stem cell expansion efficiency and purity and can serve as an in-scale high-quality skeletal muscle stem cell culture system to meet requirements for clinical research and application.