Reference molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for reference materials derived from plant raw materials. In this study, a novel reference molecule p59122, including the revealed 5' integration sequence of maize Herculex RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with 0.01 and 0.05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the reference molecule p59122 as a calibrator. All these results suggested that the developed reference molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products.