Nine chiral malonate derivatives containing 1,3,4-thiadiazole moiety (Ⅳa~i) were synthesized by one-pot asymmetric Mannich reaction using cinchona alkaloid squaramide catalyst. High yields (70%~89%) and excellent enantioselectivities [up to>99%, enantiomeric excess (e.e.)] were obtained in toluene in the presence of 10 mol% catalyst Ⅴe at 60 ℃ which were the optimum reaction conditions . The bioassay results showed that these derivatives possessed good activities against tobacco mosaic virus (TMV). Especially, chiral compound (-)-Ⅳf exhibited high curative and protective activities against TMV in vivo (50.8% and 42.6%, respectively), equivalent to that of commercially available antiviral agent riba-virin.