IL-24 and Smac are the important candidate genes for the targeting multi-genes therapy of cancer. In the construction of IL-24 and Smac dual gene expression vector, a stable and efficient linker is critical. We use three different linkers such as IETD, EEED and F2A to obtain three eukaryotic expression plasmids which are pcDNA3.1(+)-IL-24-IETD-Smac, pcDNA3.1( )-IL-24-EEED-Smac, pcDNA3.1( )-IL-24-F2A-Smac, and then combined with 5-fluorouracil (5-FU) treatment to study the cleavage efficiency of the three linker peptides. The results showed that among three IL-24-linker-Smac dual gene expression vectors, the F2A linker is superior to IETD and EEED linkers. In addition, its cleavage efficiency is positively correlated with activated caspases. Moreover, the fusion protein mediated by both IETD and EEED linker also can be cleaved,but their upstream IL-24 protein with 4 additional amino acid residues derived from IETD or EEED linker was detected with obvious degradation regulated by the ubiquitin-proteasome system. This study will provides a reference for the construction of dual-gene or muti-gene expression vector in cancer therapy.