Mesenchymal stem cells (MSCs) were used widely as seed cells in tissue engineering blood vessel construction. However, the biological characteristics difference of different generation MSCs in vitro culture is unknown, which laid a foundation for appropriate generation seeded cells selection for tissue engineering blood vessel construction. In this report, MSCs were isolated from SD rat bone marrow and identified by flow cytometry; cell growth curve test, cell surface antigen expression rate detection, cryopreservation resuscitation rate test, CD31 expression rate test, cell cycle analysis, and adhesion difference on vascular scaffold test were performed. The research results indicated that the MSCs shape was spindle and uniform with vigorous growth. CD105 and CD90 factor expression rate reached 82.5 and 84.9%, respectively, and the expression rate of CD45 was only 7.3%. The proliferation capacity of the fourth generation MSCs were more exuberant, with proliferation index as 20.3%; the cell proliferation index of the eighth generation decreased to only 9.1%. The cryopreservation resuscitation rate of the second generation and fourth generation MSCs were both higher than 80%, and the cryopreservation resuscitation rate of the eighth generation MSCs was only about 60%. After the induction for 5days, MSCs had weak CD31 expression, and with the prolonged induction time, expression increased. All generation MSCs expressed CD31 after being induced for 10days; however, the CD31 positive expression rate of the second generation, fourth generation, and sixth generation MSCs had significant difference with the eighth generation MSCs. Adhesion rate of MSCs before sixth generation was around 40%, but the adhesion rate of eighth generation MSCs was only about 27%. In all, biological characteristics of different generation MSCs existed certain differences, and especially the eighth generation MSCs aged seriously, whose cell activity decreased significantly. The researchers believed that the MSCs before the sixth generation can maintain excellent properties of MSCs, and can be used as seed cells for vascular tissue engineering.