Objective To establish a method of identification of DKO mouse model of Duchenne muscular dystrophy, and to assess the dystrophin regeneration after stem cell transplantation. Methods Heterozygous mice were mated and the resulting offspring were used to identify their genotype by SSP-PCR. The plasma creatine kinase level was measured by biochemical analyzer and histological changes in the DKO mice were analyzed using HE staining. Human umbilical cord mesenchymal stem cells were prepared and injected into the DKO mice hindlimb muscle, and dystrophin expression was detected by immunofluorescence staining at 2 months after injection. Results Mating of heterozygous mice generated three kinds of genotype offsprings, and 21.2% of the offsprings were identified as DKO genotype (285 bp). DKO mice showed dystrophic symptoms, their plasma creatine kinase level was as high as 16988.52±617.48 IU/L, and significant histological changes including diverse myocyte sizes, numerous centrally nucleated cells and connective tissue proliferation or inflammatory cells infiltration. Human dystrophin expression was detected in the DKO mouse hindlimb muscle at two months after injection of human umbilical cord mesenchymal stem cells. Conclusion DKO mouse genotype can be identified by SSP-PCR, and DKO mouse is an ideal animal model for studies of stem cell therapy for Duchenne muscular dystrophy.