Enzymatic synthesis of L-cysteine from DL-2-amino-△ 2-thiazoline-4-carboxylic acid (DL-ATC) by a mutagenised strain, Pseudomonas sp. C-25, was studied in this paper. Low-energy ion beam implantation was applied in the original strain Pseudomonas sp. B-3 and a mutant strain designated as Pseudomonas sp. C-25 was obtained, which enzyme activity achieved 2747±3.7 U/mL, an increase of 26.2% compared to that of the initial one (2175±5.1 U/mL). The components of enzyme-producing medium for Pseudomonas sp. C-25 were subsequently optimized by response surface methodology. With Plackett-Burman design (PBD) method, DL-ATC·3H2O, glycerol and beef extract were found to be the significant factors among eight tested variables that influenced the enzyme activity. Further research on the interaction among the significant elements was then conducted by means of steepest ascent, Box-Behnken design and response surface methodology. Consequently, it turned out that the optimum medium was composed of (in g/L): DL-ATC·3H2O 4.7, glycerol 16.0, beef extract 6.5, yeast extract 4.0, peptone 5.0, NaCl 3.0, MnSO4·H2O 0.06, MgSO4·7H2O 0.5. Under such conditions, the enzyme activity reached 3301±6.4 U/mL in shake culture and 3925±5.7 U/mL in 7 L fermentor, with an increase of 20.1% and 42.8% respectively, compared to that of the original medium components.