Abstract Heparinases are critical tools for the studies of highly heterogeneous heparin (HP)/heparan sulfate (HS). However, the exolytic heparinases urgently needed for the sequencing of HP/HS chains are seemingly inaccessible. Herein, a type of exolytic heparinases (exoHepases) was identified from the genomes of different bacteria. These exoHepases share almost no homology with known Hepases and prefer to digest HP rather than HS chains by sequentially releasing unsaturated disaccharides from their reducing ends. The structural study of an exoHepase (exoHep) shows that an N-terminal conserved DUF4962 superfamily domain is essential to the exolytic activity of these exoHepases, which is involved in the formation of a unique L-shaped catalytic cavity controlling the sequential digestion of substrates through electrostatic interactions. Further, several HP octasaccharides were preliminarily sequenced by using exoHep. Overall, this study fills the research gap of exoHepases and provides urgently needed tools for the structural and functional studies of HP/HS chains.