Two series of novel trifluorobutenyl derivatives of heterocyclic with convenient and efficient synthesis methods and their antitumor activity on three cell lines have been reported for the first time. The derivatives were synthesized by the nucleophilic substitution between 4-bromo-1,1,2-trifluorobutene-1-ene and commercially available nitrogen-containing heterocycles with sulfydryl or monosubstituted malononitrile. The twenty-four new compounds were characterized by (1)HNMR, (13)CNMR and HR-MS. Totally, thirty-seven compounds were evaluated for the antitumor activity on three cancer cell lines (SH-SY5Y, MCF-7 and HepG2) using conventional MTT assay. The pharmacological results indicated that the compounds 3c, 3h, 4c, 8, 9, 10 and 11 showed potent to moderate antitumor activity against three cancer cell lines, with IC50 values ranging between 0.4 μM and 41.5 μM. Even though they had less active than the reference compound taxol against MCF-7 and HepG2 lines, but they were better than the reference compound noscapine against SH-SY5Y cells, especially the compound 3h with a IC50 value of 0.4 μM.