The present disclosure discloses a crosslinked hydrogel for muscle stem cell culture and a preparation method and use thereof, and belongs to the technical field of biological food materials. The preparation method includes: dissolving collagen to prepare a solution and adding a certain amount of alginate and heparan sulfate proteoglycan for being uniformly mixed with the collagen solution; and adding ε-PL and TGase into the solution, uniformly stirring, and putting a slurry into a mold for crosslinking to obtain the hydrogel. The hydrogel is prepared by linking the collagen, the polylysine and the heparan sulfate proteoglycan using the TGase to form covalent crosslinking, and forming a compact three-dimensional “egg box” network structure through a physical electrostatic interaction between the polylysine and the alginate. The hydrogel can enhance the absorption to nutrient substances by the muscle stem cells and facilitate the growth of the muscle stem cells. The double-network crosslinked hydrogel has the potential to be a scaffold for the growth of muscle stem cells for cultured meat from stem cells.