2-Amino-4,5-dimethylthiophene-3-carbonitrile was first obtained through the modified Gewald reaction with butan-2-one, malononitrile and elemental sulfur as raw materials. Then sixteen fluorinated thieno[2,3-d]pyrimidine derivatives IIIa~IIIp were synthesized by the substitution reaction of substituted benzylamines with the key intermediate 4-chloro-5,6-dimethyl-2-(trifluoromethyl)thieno[2,3-d]pyrimidine, which was prepared directly from 2-amino-4,5-dimethylthiophene-3-carbonitrile and trifluoroacetic acid in the presence of phosphorous oxychloride via one-pot procedure. The structures of these target compounds were confirmed by 1HNMR, 13CNMR, IR, MS and elemental analysis. The crystal structure of compound IIIa was determined by X-ray single-crystal diffraction. The bioassay results suggest that the target compounds IIIa, IIIc and IIIf exhibit good in vitro antitumor activity. The target compounds IIIk~IIIp with an electron-donating substituent in the benzene ring display poor antitumor activity, but the antitumor activity is better (e.g. IIIc, IIIf and IIIi) when the meta-position of the benzene ring was substituted with an electron-withdrawing group, especially a fluorine atom. The half inhibitory concentration (IC50) values of compound IIIa against MCF-7 and HepG2 cells were 2.01 μmol/L and 2.44 μmol/L, respectively, while the IC50 values of IIIc against MCF-7 and HepG2 cells were 1.44 μmol/L and 1.47 μmol/L, respectively. Both of them indicate much better antitumor activity than the control group Gefitinib.