Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China. Radiotherapy and chemotherapy are the main therapy methods for NPC. To enhance the specific antitumor effect, a novel vector with radiosensitivity and tumor specificity was constructed in this study, which enables the reduction of dosage of radiation and chemotherapeutic drugs due to its double killing effect. Four DNA elements, Egr-1 promoter, Cytosine deaminase (CD) gene, hTERT promoter, Survivin antisense oligonucleotides were amplified and constructed in pcDNA3.1 vector. CD and Survivin gene expression in CNE-2 cells were detected by RT-PCR. High performance liquid chromatography (HPLC) was employed to determine the transformation from the prodrugs 5-FC to 5-FU. Hoechst33258 staining of the nuclei and methylthiazolyl tetrazolium(MTT) assay were applied to detect apoptosis and cell survivability, respectively. In addition, the anti-tumor effects were examined in vivo by injecting cells with different vectors into nude mice. Our results revealed a notable killing effect of combined treatment with 5-FC and radiation on CNE-2 cells transfected with vectors in vitro. This effect was especially notable on pEC-TS transferred cells, which showed 57% of cells were killed. In vivo, an obvious suppression of tumor was displayed in pEC-TS group, which was significantly different from other groups (p < 0.05). Consequently, this expression cassette may have a great therapeutic potential for the treatment of NPC.