A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2-630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2-630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P-3, P-4) and TaqMan fluorescence probe (P3-14) were designed based on the R2-630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F-1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 A degrees C and the Mg2+ concentration is 3.5 mmol/L.