Thirteen fluorinated thieno[2,3-d]pyrimidine derivatives were synthesized in the yields of 69 ~ 83% by the SNAr reaction of substituted benzylamines with 4-chloro-2-trifluoromethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyridine, which was prepared directly from 2-amino-3-carbo-nitrile-tetrahydro[4,5]benzothieno[2,3-d]pyrimidine and trifluoroacetic acid (TFA) in the presence of phosphorous oxychloride via one-pot procedure. While 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile was first obtained according to the modified Gewald reaction with cyclohexanone, malononitrile and elemental sulfur as staring materials. The structures of these title compounds were confirmed by 1H NMR, IR, MS and elemental analysis. The crystal structure of compound IIIa was determined by single-crystal X-ray diffraction. The results show that this compound crystal belongs to monoclinic crystal system, space group C2/c with a = 2.6959(4) nm, b = 0.77245(13) nm, c = 1.6885(3) nm, α = 90? β = 109.320(2)? γ = 90? V = 3.3182(9) nm3, Z = 8, Dc = 1.455 Mg?m?3, μ = 0.23 mm?1, F(000) = 1504, R1 = 0.0579, wR2 = 0.1604. The preliminary bioassay results suggested that some of the title compounds exhibit good in vitro antitumor activity against HepG2 and MCF-7, especially the compounds IIIc, IIIf and IIIj exhibited higher inhibitory activity than the positive control gefitinib.