cis-Epoxysuccinate hydrolase (CESH) is efficient in catalyzing conversion of cis-epoxysuccinate into l-(+)-tartaric acid, and the enzyme has been successfully applied in industry. However, low thermostability limits its use in large-scale applications. To improve the stability of CESH, we conducted directed evolution and used strategies belonging to semi-rational redesign. Mutant 1X-1 (Q122R) was harvested from directed evolution. Its half-life at 50°C (t1/2, 50°C) increased from 8.5min to 31.6min, and the T5015 (temperature at which the activity of enzyme decreased by 50% in 15min) increased from 44.0°C to 49.5°C. Substitutions F26V and I83R predicted by multiple sequence alignments were introduced into mutant 1X-1, and the t1/2, 50°C and T5015 of mutant 3X (Q122R, F26V and I83R) increased to 170.8min and 55.4°C. Simulated mutation based on in silico structural modelling was used in constructing mutant 5X (Q122R, F26V, I83R, D8K and S90R), for which the t1/2, 50°C and T5015 increased to 237.1min and 62.4°C, respectively. Site-saturated mutagenesis was employed on amino acid residues Asp-8, Phe-26, Ile-83, Ser-90, and Gln-122 to maximize the thermostability of mutant 5X. Mutant 5X-1 (Q122R, F26W, I83R, D8K and S90R) was isolated; its t1/2,50°C increased to 293.2min, 34.5-fold that of the wild-type enzyme and the T5015 increased to 64.8°C. Moreover, the effective working ranges of pH of mutant 5X-1 extended to 5.0–10.0 from 8.0–9.0 for the wild-type enzyme.