Objective To screen the cells and construct primers to be used for RNA interference experiment of ERβ gene in Beagles.Methods Three pairs of primers were designed according to the amino terminal and DNA binding area of ERβ gene in Beagles, and the best primers were screened as the primers to be used to detect RNA interference effect by positive plasmid, and the cDNA from 293T, Hela and Vero cells was used to validate the specificity of the primers. The existence of homo ERβ gene was also tested in the three cell types. Results After experiments repeated for three times, the results showed that they had high amplification effect and there was no nonspecific amplification by the C36684 primers. The amplification results of cDNA from 293T, Hela and Vero cells also showed that there was no nonspecific amplification by the primers, but there was homo ERβ gene expression in 293T cells. Conclusion The C36684 primer pair is selected for detecting RNA interference effect, and Hela cells are selected as the tool for RNA interference experiment.