The invention discloses a canine distemper virus (CDV) sensitive cell line and an establishment method and application thereof, and belongs to the technical field of biology. According to the hundestaupe virus sensitive cell line, lentivirus four-plasmid packaging system is adopted to obtain a strain of stable cell line madin-darby canine kidney-cell isolate (MDCK-CSL) which expresses canine source signal lymphocyte activating molecules (SLAM), wherein the conservation number of strains is CGMCC No. 5881. The comparison of the propagation conditions of a CDV standard strain on the MDCK-CSL and MDCK cells indicates that after the CDV-Snyder Hill standard strain infects the MDCK-CSL cell line for 24 hours, cytopathy of the fusion, rounding contraction, obvious death and the like of cells can be observed; and the MDCK cells infected for 6 days continuously grow slowly, and the cytopathy does not occur. The CDV sensitive cell line MDCK-CSL provides a technical platform for the separation of CDV wild strains and the complete study of biological properties of the CDV wild strains, and also establishes a foundation for the prevention and control of canine distemper.