Objective This paper reports a modified method for the isolation of rat thoracic aortic vascular smooth muscle cells based on combined enzyme double digestion. Methods An enzyme mixture containing collagenase Ⅱ, soybean trypsin inhibitor and elastase was prepared and used to remove the aortic tunica intima and tunica adventitia, and then the tunica media was subjected to second digestion using the same enzyme mixture and to isolate vascular smooth muscle cells. Results The isolated VSMCs were cultured in vitro and the growing cells had an elongated spindle-shape, reached confluence within a week, and displaying a typical "hill-and-valley" pattern. After 1 week, the cells were passaged. Contractile smooth muscle markers(α-SMA, myosin-Ⅱ and β-tubulin) were highly expressed in the isolated cells. More than 90% of the cells significantly expressed alpha smooth muscle actin and myosin-Ⅱ. Conclusions Themethod established in this study has advantages of simple and easy to operate, and good reproducibility, with a high activity and purity of the separated cells. It can ensure to obtain a large amount of contraction-type vascular smooth muscle cells within a short time.