Ultra-low molecular weight heparins (ULMWHs) are being used as anticoagulant drugs that exhibit a low risk, long lifetime and high efficacy. However, a robust and cost-effective purification methodology to isolate ULMWHs from naturally occurring heparins (HPs) is lacking. Vaccinia virus envelope protein A27 and a truncated variant (sA27-aa) bind specifically to HPs for virus/cell attachment. Here, we reported that using a high-affinity bioengineered sA27-aa for heparinase depolymerisation enables extraction of size-specific oligosaccharides, such as tetra-, hexa- and octa-saccharides (HP-4, -6 and − 8, respectively), with high purity and yield. Molecular interactions of the HP-4•viral protein complex and HP-4 sugar composition were resolved by mass and NMR, with data from both spectrometry in good agreement. As demonstrated by in vivo and in vitro bioassays, HP-4 possesses superior antithrombotic efficacy, stable properties and a low bleeding risk. Our methodology facilitates unprecedented extraction of unique ULMWHs that could benefit patients suffering from thrombosis.