Hirudin is the most potent natural inhibitor of thrombin and a powerful anticoagulant. Large-scale production of recombinant hirudin is desirable for therapy. In this study, the gene encoding hirudin variant III was redesigned and synthesized by using Bacillus subtilis preferred codons, and the recombinant hirudin variant III (rHV3) was overexpressed in B. subtilis DB403 with strong anticoagulation activity for the first time. The hirudin activity from the supernatant of culture with optimized expression conditions could reach 210 ATU/ml. The protein in culture supernatant was precipitated by trichloroacetic acid, then desalted by ultrafiltration and purified by anion exchange chromatography. Strong anion Q F.F. performed better than weak anion DEAE F.F. The proper pH and conductivity was determined at pH 8 and 6 ms/cm, respectively. The maximum applied sample was 240 ATU/ml to medium of strong anion Q F.F. This optimized procedure was employed in strong anion exchange HiPrep 16/10Q with the 90% recovery rate and 70.2% purity. After gel filtration, the purity of rHV3 checked by HPLC could reach 95.1%, and the recovery rate was 93% for this step. The purified recombinant rHV3 showed a single band in SDS-PAGE. The rHV3 was stable at 100 °C and acidity condition, but was unstable under the condition of both heating and alkalinity. In conclusion, theses studies suggests that B.subtilis might be useful for the production of biologically active medicine peptides in secretion facilitating purification procedures, and that this isolation method was suitable for scale-up purification process at a low cost.