Abstract: A novel strain named SK23.001 was isolated from soil in Watermelon canopy and it was identified as Enterococcus faecalis. In order to enlarge the yield of citrulline, the fermentation of Enterococcus faecalis SK23.001 were optimalized. The optimal vaccination age and culture time was 12h and 16h, respectively. The optimum fermentation conditions by single -factor experiment and orthogonal experiment were determined:carbon source sucrose (0.5%) , nitrogen source soya peptone and ammonium chloride, inoculation volume 3%, liquid medium volume 50mL/250mL, pH6.0, temperature 37℃ . The product was analyzed by HPLC and the 83.06g/L of citrulline transformed from 100g/L arginine could be attained after 6h.