There is an urgent clinical need of tissue-engineering (TE) vascular grafts, so this study was for developing a fast and simple way of producing TE vascular scaffold. The TE vascular scaffold was prepared with pepsin, DNase and RNase enzymatic decellularization and crosslinked with 0.1, 1, 5% glutaraldehyde (GA), respectively. The samples were underwent analyses of burst pressure; suture strength; cytotoxicity; enzymatic degradation in vitro; degradation in vivo; rehydration; biocompatibilities detected with hematoxylin and eosin (H&E), scan electron microscope, immunohistochemistry both in vivo and in vitro; macrophage infiltration and calcification using Von Kossa staining. After being decellularized the scaffold had a complete removal of cellular components, an intact collagen structure. The burst pressure and suture strength were similar to native artery. 0.1% GA crosslinked scaffold showed less cytotoxicity than 1 and 5% GA groups (P < 0.05) and was resistance to enzymatic degradation in vitro. Once being implanted, 0.1% GA group was resistant to degradation and formed endothelium, smooth muscle and adventitia with few macrophages infiltration. However, there appeared calcification in implants compared with that in native artery. This study demonstrated that DVPs producing methods by enzymatic decellularizing and crosslinking with 0.1% GA could be used for clinical TE vascular graft manufacture.