A chemoenzymatic approach, mimicking the biosynthetic pathway of heparin and heparan sulfate (HS), has been well developed to prepare a series of structurally well-defined heparin oligosaccharides with excellent anticoagulant activity in good overall yields. The current chemoenzymatic synthesis typically begins with an unnatural glycosyl acceptor, p-nitrophenyl glucuronide (GlcA-PNP), which is convenient for detection recovery and purification, although it affords heparin molecules with undesirable structure characteristics. Herein, we describe a facile chemoenzymatic strategy assisted by the specific cleavage of heparinase III for the highly efficient synthesis of an unmodified heparin heptasaccharide which demonstrated potent anticoagulant activity in vitro and commensurate pharmacokinetic profiles with fondaparinux. This successful generic strategy is applicable to the scalable synthesis of diverse HS/heparin molecules with completely natural structural features as promising therapeutic agents.