Abstract: The use of RNA interference (RNAi) is becoming a routine method of scientific discovery and treatment of human diseases. However, its application is hindered by adverse effects, especially the off-target effects. In this work, we established an shRNA test platform based on the plasmid to test the silencing ability of short hairpin RNA (shRNA) targeting survivin gene designed according to shRNA online software and then screened out two shRNAs with excellent silencing ability. The off-target effects were measured by testing the silencing ability of two selected shRNAs against targets with single-nucleotide mutations at different locations. Based on our understanding of the 3′ tail function of microRNA (miRNA) and small interfering RNA (siRNA), we propose a method to reduce the off-target effect of short hairpin RNA by shortening the complementary length of the 3′ tail and target sequence of siRNA only. This method can reduce the off-target effect of shRNA without damaging the silencing efficiency of shRNA gene, so as to effectively improve the specificity of shRNA gene silencing. Compared to previous studies of the weak base pairing of “seed” and 3′ regions to reduce the off-target effect, our method is simpler and more efficient, without being limited by the sequence of 3′ regions of the antisense strand. This method of reducing the off-target effects provides new ideas for the design of shRNA and siRNA, simplifies the sequence restriction in the design of shRNA and siRNA drugs to a certain extent, and broadens their use and prospects as therapeutic and diagnostic tools in medical treatment.