Objective  To construct a co-expressing lentiviral vector of preoptic regulatory factor-2 (Porf-2) and green fluorescent protein (GFP), package the lentiviruses, and transfect neural cells in vitro. Methods  Primers of Porf-2 were designed according to the gene information of GenBank. Gene fragments of Porf-2 were amplified by polymerase chain reaction (PCR). By using the gene recombinant technology, Porf-2 was double digested and cloned to pLVX-IRES-ZsGreen1 vector. Recombined plasmids were identified by enzyme digestion and DNA sequencing. The 293T cells were transfected with constructed plasmids, packaging system VSVG, and Δ89 plasmids. Lentiviruses were packaged and the titer was determined. Results  Enzyme digestion and DNA sequencing confirmed that the gene recombinant lentiviral vector of pLvx-Porf-2-IRES-ZsGreen1 had been successfully constructed. After NG108, primary hippocampal neurons, and neural stem cells were transfected by the packaged lentiviruses, a large number of 293T cells with green fluorescence were observed. Western blotting confirmed that Porf-2 was successfully over-expressed. Conclusion  The co-expressing lentiviral vector of Porf-2 and GFP is successfully constructed; lentiviruses are packaged; and NG108, primary hippocampal neurons, and neural stem cells are transfected.