Recent years, people are paying more and more attention to amniotic membrane tissue which has a rich resource and no ethical restrictions. Canine amniotic mesenchymal stem cells (cAMSCs) were collected by enzyme digestion method from canine amniotic membrane tissues. The cell morphology was continuously observed at different passages and cell growth curves of passage 3 and passage 9 were drawed. Stem cell related proteins and genes were identified by immunofluorescence and RT-PCR. The multiple differentiation potential of cAMSCs was identified by osteogenesis and adipogenesis. This study showed that the cAMSCs was long fusiform and could be continuously subcultured in vitro. The immunofluorescence results showed that Vimentin and SSEA-4 were positively expressed. The RT-PCR results showed that stem cell related genes OCT4, SOX2 and NANOG were positively expressed too. The cAMSCs could be induced to osteoblasts and adipocytes in vitro. The markers associated with osteogenesis and adipogenesis, such as COL1A1 and LPL, were positively expressed after differentiation. The results showed that the cAMSCs could be successfully isolated and cultured in vitro. Stem cell related proteins and genes were positively expressed. And the cAMSCs has the multiple differentiation potential. This study showed that cAMSCs could be a rich source of stem cells in veterinary. Furthermore, cAMSCs may be useful as a cell therapy application for veterinary regenerative medicine.