The radiosensitizer compounds are used in order to increase the efficiency of radiotherapy. Different agents have been evaluated for radiosensitizer effects. 2,6-bis-(2,6 dichlorobenzylidene) cyclohexanone is a α, β unsaturated ketone derivatives. Conjugated unsaturated ketones can be readily reacted with thiol groups which are not found in nucleic acid. Theoretically, the genotoxicity side effect should not be observed from these agents. The suggested conjugated unsaturated ketone was synthesized from the reaction of cyclohexanone and 2, 6 dichlorobenzaldehyde in the presence of catalytic amount of NaOH. The 1HNMR analysis demonstrated that the desired compound could be successfully prepared with the appropriate yield. The cytotoxicity of prepared compound and 2 Gy ionizing irradiation was investigated on HT 29 cell line which was resistant to 2 Gy radiation due to MTT assay. The outcome of this approach indicated that the prepared selective thiol alkylator with radiation could significantly increase the mortality of treated cells versus to the cells were only treated with the prepared compound. Therefore, the conjugated unsaturated ketone can open the new field for the preparation of new radiosensitizer agents.