Described is a crosslinked hydrogel for muscle stem cell culture and a preparation method and use thereof. The preparation method includes: dissolving collagen to prepare a solution and adding alginate and heparan sulfate proteoglycan and uniformly mixing with the collagen solution; 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.