Eight fluorinated pyrazolo[3,4-d]pyrimidine derivatives containing 1,3,4-thiadiazole were synthesized in the yields of 79 ~ 87% by SNAr reaction of 5-aryl-2-mercapto-1,3,4-thiadiazoles with 4-chloro-1-phenyl-6-trifluoromethyl-1H-pyrazolo[3,4-d]pyrimidine, which were efficiently prepared from 5-amino-4-cyano-1-phenyl-1H-pyrazole, trifluoroacetic acid and trichlorophosphate by one-pot procedure in 61% yield. The structures of the products were confirmed by IR, 1H NMR, MS and elemental analysis. The primary antibacterial activity of these compounds against two bacteria (Staphylococcus aureus and Escherichia coli) was screened. The results indicated that the title compounds exhibit antibacterial activity against Staphylococcus aureus, and compounds IVb and IVh have relatively good bacteriostatic effect.