Decellularization is a promising approach in tissue engineering to generate small-diameter blood vessels. However, some challenges still exist. We performed two decellularization phases to develop an optimal decellularized scaffold and analyze the relationship between the extracellular matrix (ECM) composition and mechanical properties. In decellularization phase I, we tested sodium dodecylsulfate (SDS), Triton X-100 (TX100) and trypsin at different concentrations and exposure times. In decellularization phase II, we systematically compared five combined decellularization protocols based on the results of phase I to identify the optimal method. These protocols tested cell removal, ECM preservation, mechanical properties, and residual cytotoxicity. We further immobilized heparin to optimal decellularized scaffolds and determined its anticoagulant activity and mechanical properties. The combined decellularization protocol comprising treatment with 0.5% SDS followed by 1% TX100 could completely remove the cellular contents and preserve the mechanical properties and ECM architecture better. In addition, the heparinized decellularized scaffolds not only had sustained anticoagulant activity, but also similar mechanical properties to native vessels. In conclusion, heparinized decellularized scaffolds represent a promising direction for small-diameter vascular grafts, although further in vivo studies are needed. © 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group.