To explore the role of miR-33 in the regulation of fat metabolism by sterol regulatory element binding protein 1(SREBP-1), the dual-luciferase reporter assay vector containing SREBP-1 gene 3'-untranslated region of Ctenopharyngodon idella has been constructed. The 3'-UTR of SREBP-1 with miR-33 combining site was first amplified(with the length of 379 bp) and transfected into competent cells(DH5α) after being recombined with dual-luciferase reporter assay vector(pmirGLO). Screening and identifying with XbaI and SacI to digest the recombinant vector were then conducted. Finally, the relationship between miR-33 and the 3'-UTR of SREBP-1 was analysed by the co-transfection of the recombinant vector and miR-33 mimics into L8824 cells. The electrophoretogram of PCR products, the restriction map of recombinant vector, and the result of gene sequencing comfirmed that the dual-luciferase reporter assay vector containing 3'-UTR of SREBP-1 was successfully constructed. In addition, the activity of luciferase expressed by transfected L8824 cells was detected and the expression level of the control was significantly higher than that of the co-transfected group including the recombinant vector and miRNA-33 mimics. The results suggested SREBP-1 is the target gene of miR-33. By binding to the 3'-UTR region of SREBP-1, miR-33 plays an important role in the regulation of post-transcription of SREBP-1.