Abstract： Objective To construct an eukaryotic expression vector for shRNA targeting WT1 gene.Methods Firstly, one site targeting human WT1 gene in gene bank was selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed. Secondly,a pair of oligonucleotides fragments were synthesized and annealed to double strand. Thirdly, the double strand was cloned into plasmid vector pGenesil-1. Finally, the recombinant plasmid was identified by restriction enzyme digestion and sequencing analysis. Results Enzyme cutting, and sequencing analysis have confirmed that the construction was successful. Conclusion WT1shRNA is constructed which is the fundament to further research of WT1 gene.