A process for producing antithrombin (AT) prepared through genetic engineering technique； and a method of purification thereof. In particular, a recombinant antithrombin (rAT) having been improved with respect to production volume and specific activity can be produced by culturing animal cells capable of production of (AT) transformed by genetic engineering technique under conditions of a combination of 6.9 or below pH, 4 mM or more glutamic acid and 35°C or below. Further, increasing of (rAT) specific activity, high-degree purification and turbidity elimination can be accomplished by subjecting a culture supernatant containing rAT or the like to treatment with immobilized heparin, solvent exchange, anion exchanger treatment, polymer removal, filtration through a porous membrane and treatment at 7.5 or higher pH.
