More and more stacked GMOs have been developed for more improved functional properties and/or a stronger intended characteristic, such as antipest, improved product efficiency etc. Bt11 x GA21 is a new kind of stacked GM maize developed by Monsanto Company. Since there are no unique flanking sequences in stacked GMOs, up to now, no appropriate method has been reported to accurately detect them. In this passage, a novel universal primer multiplex PCR (UP-M-PCR) was developed and applied as a rapid screening method for the simultaneous detection of five target sequences (NOS, 35S, Bt11 event, GA21 event, and IVR) in maize Bt11 x GA21. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers. What's more, it got a high specificity and had a detection limit of 0.1% (approximates to 38 haploid genome copies). Furthermore, real-time PCR combined with multivariate statistical analysis was used for accurate quantification of stacked GM maize Bt11 x GA21 in 100% GM maize mixture (Bt11 x GA21, Bt11 and GA21). Detection results showed that this method could accurately validate the content of Bt11, GA21 and Bt11 x GA21 in 100% GM mixture with a detection limit of 0.5% (approximates to 200 haploid genome copies) and a low relative standard deviation <5%. All the data proved that this method may be widely applied in event-specific detection of other stacked GMOs in GM-mixture.