Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenases (HHDHs) is of great value due to the 100% theoretical yield and high enantioselectivity. The vital problem in the asymmetric synthesis is to prepare optically pure ECH. In this study, key amino acid residues located at halide ion channels of HheC (P175S/W249P) (HheC(PS)) were modified to regulate the kinetic parameters. HheC(PS) I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed. The catalytically efficiencies (k(cat)/K-m) of the three mutants exhibited 0.38-, 0.23-and 0.23-fold decrease toward (S)-ECH and the reverse reaction was significantly inhibited. As the results, (S)-ECH was synthesized with> 99% enantiomeric excess (e.e.) and 63.42%, 67.08% and 57.01% yields, respectively, under 20mM 1,3-DCP as substrate. To our knowledge, this is the first investigation of the molecule kinetic modification of HHDHs and also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDHs.