Integrons can capture and express foreign gene cassettes through site-specific recombination and are important genetic elements in spreading antibiotic resistance genes among bacteria. We have developed a two-dimensional PCR technology (2D-PCR) based on the base quenching probe technology in detecting three major integrons at the same time. The minimum detection limits were evaluated by detecting three plasmids each harboring different types of integron with different concentrations. The specificity of this method was evaluated by screening and typing three major types of integrons in 105 clinical Proteus isolates, and the results were compared with those of traditional PCR. Results indicated that the melting temperature (Tm) difference of the three genes was about 10 °C and was very easy to be distinguished. The minimum detection limits of intI1, intI2 and intI3 were all below 102 copies/μl. The detection results of clinical isolates were consistent with those of traditional PCR. This developed rapid, economic and high-throughput 2D-PCR based method can detect three main classes of integron at the same reaction, and can be applied to clinical isolates in large-scale integron screening and typing.© 2022 Published by Elsevier Ltd.