Here, we established a strategy (MPT-Cas12a/13a) that combined CRISPR/Cas12a and Cas13a for simultaneously detecting CaMV35S and T-nos based on multiplex PCR (M-PCR) and transcription. It realized a simultaneous detection mode with different signals in the same space. The MPT-Cas12a/13a had excellent sensitivity with the limit of detection as low as 11 copies of T-nos and 13 copies of CaMV35S and it had outstanding specificity and anti-interference ability in actual sample analysis. Therefore, it is a potential candidate in the detection of GM crops.