Ten fluorinated thieno[2,3-d]pyrimidine derivatives containing 1,3,4-thiadiazole were synthesized in the yields of 64 ~ 75% by SNAr reaction of 2-amino-5-(substituted benzylthio)- 1,3,4-thiadiazoles with 4-chloro-2-trifluoromethylthieno[2,3-d]pyrimidine, which were efficiently prepared from 2-aminothiophene-3-carbonitrile, trifluoroacetic acid and phosphorus oxychloride by one-pot procedure in 60% yield. The structures of the products were confirmed by IR, 1H NMR, MS and elemental analysis. The in vitro antitumor activity of these compounds against HepG2 and BGC-823 cell lines was evaluated by MTT assay. The results showed that compounds IVc and IVf possess higher antitumor activity than the positive control gefitinib.