A practical method was established to resolute L-Serine into L-Tryptophan via Tryptophanase, using D,L-Serine as the substrate. D-Serine could be obtained after isolation and purification. Response surface methodology was used to optimize the conditions of the enzymatic resolution. And the optimum conditions were: 45℃, pH 8.0, 18 hours, 30 g/L of substrate concentration and 6 g/L of Tryptophanase concentration. After twice similar reactions, the total conversion rate of L-Serine could be up to 95.4% .The products were separated and purified on NKA-II macroporous absorption resin, 001×7 ion-exchange resin and recrystallized at the end. The purity of the products was 99.4%.The optical rotation of D-Serine was -15.3°(C=10,2N HCl) and the total separation recovery of the products was 66.6 % .