Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.