A novel method for duplex probes is designed to simulate the TaqMan probe during polymerase chain reaction(PCR). In this method, two partly complementary single-labelled oligonucleotide probes labelled with afluorophore or a quencher, respectively, are used. At lower temperature the two probes can bind to each otherand form a mismatched duplex, in which the fluorophore and quencher are in close proximity and the sameenergy transfer mechanism as in molecular beacons may occur between them; thus, a quenching efficiencybetter than conventional TaqMan probes is acquired. In the anneal-extend step of PCR, one single-labelledprobe hybridises to the predetermined target and is cleaved by Taq DNA polymerase. Increased fluorescentsignal can be observed at lower temperature. The fluorescent data analysis demonstrated that a significantlyhigher level of fluorescent signal and hence higher sensitivity of detection is obtainable using our duplex probesin place of conventional TaqMan probes. Combined with real-time PCR instruments, the assay can be used toquantify the input target molecules and the dynamic linear range is of at least six orders of magnitude.