[Objectives] To establish a PCR method to detect SV5 in biological material from monkeys. [Methods] We have designed a pair of primers matched with SH gene of SV5 for PCR and sequenced the product fragments with BLAST soft to identify the homology and conformed by with AccIII restriction digestion. we also designed nested primers to improve the sensitivity of the PCR. The PCR technique was used to detect our cells and sera from Rhesus monkeys. [Results] The PCR product was proved to be the frangment of the SH gene of SV5, and the PCR product was digested with the AccIII which is a specific site of the gene fragment. The sensibility of the examination was improved by nested PCR. All samples from monkeys were detected to be negative using this PCR method. [Conclusions] We established the PCR method to detect SV5.