Optical pure lactic acid is a useful chiral intermediate in the synthesis of chiral drugs and materials engineering. For the purpose of environmental-friendly and high stereoselectivity, enzymes were used to resolve racemic mixtures. In this study, we cloned and expressed L-2-haloacid dehalogenase (ST2570) from thermophilic Archaea Sulfolobus tokodaii in Escherichia coli BL21(DE3). The recombinant enzyme was purified to a single band using heat treatment and Ni2+-NTA affinity chromatography. The optimum temperature and pH value of ST2570 were 70 degrees C and 9.5, respectively. The specific activity of purified ST2570 for the hydrolysis of 2-chloropropionic acid was 2963.33 U/mg. For the high enatioselectivity of ST2570, only L-2-chloropropionic acid can be hydrolyzed to produce D-lactic acid during the reaction. With the optimization, the best conditions to produce D-lactic acid were pH of 9.5, temperature of 60 degrees C, 3 mol/L of buffer, 0.5 mol/L of 2-chloropropionic acid and 3x10(4) U/L of ST2570.