ObjectiveTo investigate the regulatory effects of two kinds of SV40 poly(A) signals on the upstream gene expression in three cell lines, so as to provide theoretical evidence for selection of poly(A) of vectors. MethodsA dual luciferase reporter vector Dual-Luc was constructed, and two SV40 poly(A) signal sequences were inserted in the downstream of the R-Luc gene separately. Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293, L-02, and HeLa cells. The relative quantities of the target gene (F-Luc) to control gene (R-Luc) were measured by Dual-GloTM Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). ResultsThe two Dual-Luciferase vectors (Dual-Luc2 and Dual-Luc3) were successfully constructed. Dual-GloTM Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells（P>0.05）, 6.16±0.39 and 3.83±0.39 in L-02 cells（P<0.05）, and 1.21±0.10 and 0.66±0.02 in HeLa cells（P<0.05）, respectively. The results of RT-PCR were consistent with those of luciferase assay. ConclusionThe two kinds of SV40 poly(A) signals have different regulatory effects on the upstream gene expression in different cell lines. SV40 poly(A) signals regulate the upstream gene expression at the transcriptional stage.