In this study, forty-four chiral triazole derivatives have been prepared via asymmetric synthesis, and which has been successfully characterized by typical spectroscopic techniques including 1H NMR, 13C NMR, EI-MS, elemental analysis and optical rotations. Their in vitro antiviral activities against EV71 and CVB3 were fully investigated in cell-based assays. It was observed that 13 synthetic triazole derivatives inhibited the CPE of EV71 on RD cells, with EC50S in the 5.3-15.9 μg/ml range and corresponding SIs of 4.0-27.6, while 17 triazole derivatives showed antiviral activities against CVB3, with EC50S in the 4.7-15.1 μg/ml range and the corresponding SIs of 3.7-14.5. In addition, in some cases, the respective enantiomers showed significantly selective inhibitory effect against EV71, most notably for the enantiomers 9(R) and 10(S), 42(R) and 43(S), which presented an obvious activity difference. The most potential molecules are the compounds 10 and 43 with S-configuration, and which exhibit good SI values compared with the control Ribavirin.