The invention belongs to the technical field of animal gene engineering, relating to clone of cattle longisimus dorsi tenderness related CAPN1 gene segment and an application thereof in cattle marker assisting selection. The obtained CAPN1 gene segment has the nucleotide sequence length of 520bp and comprises full fourteenth exons. A base mutation from A to G exists at the 166bp of the segment, which results in the generation of PsuI-RFLP restriction enzyme polymorphism. The mutation site is taken as a molecular marker, the restriction enzyme PsuI is used for detecting the mutation site, and association analysis can be carried out on the detection result and the cattle longisimus dorsi tenderness. Analysis result shows that striking difference exists among the longisimus dorsi tenderness of different genotype individuals, thus providing a theoretical basis for the seed selection and breeding of cattle. The invention is characterized by obtaining the CAPN1 gene part segment related to the cattle longisimus dorsi tenderness and providing the molecular marker and the application for the marker assisting selection of cattle by using a PCR-RFLP method to carry out polymorphism analysis on the segment.