Objective To construct and identify RNAi lentiviral expression vectors targeting human neurogenic differentiation gene (NeuroD). Methods The oligo DNA sequences of 4 pairs of shRNA, named as NeuroD1, NeuroD2, NeuroD3, and NeuroD4, were designed according to NeuroD gene sequence (GenBank:NM_002500). The single strand of oligo DNA was annealed to form double strand DNA, and then was cloned into the empty plasmid pcDNA^TM 6.2-GW/EmGFP-miR. Four interference plasmids were constructed and transformed into competent cells DH5α. Interference carrier was transiently transfected into target cells and the interference effect against target genes was detected by real-time PCR．The interference plasmid pcDNA^TM 6.2-GW/EmGFP-miR-NeuroD1 was linked to lentiviral destination vector pLenti6.3/V5-DEST to form the lentiviral expression vector pLenti6.3-EGFP-NeuroD1-miR. Constructed lentiviral vector carrier and packaging plasmids (Packaging Mix) were cotransfected into 293T cells, and followed by packaging virus, collecting the virus stock solution, ultra-centrifugating, condensing, and detecting the titer．PCR method was used to identify the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results Four interference plasmids for target gene were successfully constructed. The sequences of expression vector pcDNA^TM 6.2-GW/EmGFP-miR-NeuroD1/2/3/4 were proven correct using sequencing method. miR-NeuroD1 sequence showed the best silencing effect after transfected into 293T cells (P<0.01). Restriction endonuclease and PCR analysis confirmed that the pcDNA^TM 6.2-GW/EmGFP-miR-NeuroD1 was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring NeuroD1 shRNA was 1.18×108 ifu/mL. Conclusion The recombinant lentivirus pLenti6.3-EGFP-NeuroD1-miR has been constructed successfully, which lays a foundation for future study of NeuroD function.