Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with the chaperone expression system (pGro7) was constructed and transferred to E. coli BL21 (DE3) to co-express recombinant strain E. toll BL21 (DE3)/pET-28a-3-OST-1 + pGro7. The soluble expression of 3-OST-1 was significantly improved in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/mL and having a specific activity of 0.83 U/mg. N-sulfo, N-acetylheparosan (NSNAH) was modified by the recombinant expressed 3-OST-1 and the product was confirmed by H-1 NMR showing the sulfo group was successfully transferred to NSNAH.