RNA interference (RNAi) provides a powerful tool to silence genes in a sequence-specific manner in a variety of systems. However, not all sequences are effective in the RNAi-mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and beta-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning.