Objective To construct a muscle-specific Cas9-tracking expression vector to examine myoblast differentiation and establish a muscle-specific Cas9-expressing mouse model. Methods The muscle-specific promoter SP was synthesized to replace the CMV promoter in the PX459 vector. The editing efficiency of the recombinant vector in C2C12 cells was assessed by XbaI and T7E1 digestion. A DsRed fluorescent reporter protein was linked through homologous recombination to construct a muscle-specific Cas9-tracking expression vector (PX459-Rosa26-SP-DsRed). The SP-Cas9-DsRed fragment was connected between the left and right homologous arms of the Rosa26 site to construct a muscle-specific Cas9-tracking expression recombinant vector. The vector was cotransfected with PX459-Rosa26 into C2C12 cells that were selected by puromycin and fluorescence was observed. DNA was extracted for PCR and sequencing to detect integration of the vector in C2C12 cells. Results Enzyme digestion and sequencing showed that PX459-Rosa26-SP and Donor-Cas9-SP-DsRed were constructed successfully. Enzyme digestion showed that the editing efficiency of PX459- Rosa26-SP was 18. 38%. The transfected C2C12 cells emitted red fluorescence, indicating that Donor-Cas9-SP-DsRed was specifically expressed in muscle cells. PCR and sequencing showed that Donor-Cas9-SP-DsRed was successfully integrated at the Rosa26 site in C2C12 cells. Conclusions A muscle-specific Cas9-tracking expression vector was constructed successfully, which provides the basis to prepare a muscle-specific Cas9-expressing mouse model and new research ideas for gene therapy of muscle-related genetic diseases.