Aims One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large‐scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. Methods and Results The 6‐O‐sulfotransferase isoform‐3 (6‐OST‐3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria–Bertani medium in shake flasks and inducing with isopropyl β‐D‐1‐thiogalactopyranoside at an optical density of 0·6–0·8. The production of larger amounts of 6‐OST‐3 required fed‐batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5–20 mg g‐cell‐dry weight⁻¹) of active 6‐OST‐3 with excellent productivity (2–5 mg l⁻¹ h⁻¹). Conclusions The production of 6‐OST‐3 in a fed‐batch fermentation on an inexpensive carbon source has been demonstrated. Significance and Impact of the Study The ability to scale‐up the production of heparin biosynthetic enzymes, such as 6‐OST‐3, is critical for scaling‐up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal‐sourced anticoagulant product currently on the market.