Bone marrow mesenchymal stem cells (BMMSCs) were separated by density gradient centrifugation and purified by different adherence speed. BMMSC was identified by flow cytometry analysis and laser confocal microscopy. Cell surface antigen (CSA) CD29, CD44, CD105, CD166 were detected positive expression and CD14, CD34, CD45 were not positive. The positive expression of the nestin, NST, and weakly positive expression of Oct-4 were confirmed by RT-PCR. The cells were verified as the BMMSC by the expression of the CSA and the genes. Using density gradient separation and different adherence speed could harvest high quality BMMSC. This is a simple and reliable method to obtain BMMSC as stem cell used in cell and tissue engineering research.