In order to establish a multi-PCR detection method for the simultaneous detection of class 1 integron (Int1), class 4 integron (SXT) and insertion sequence common region 1(ISCR1), three pairs of specific primers were designed using PrimerPlex 2.61 based on the gene sequence of int1, int_SXT and ISCR1 in GenBank. The multiplex PCR system was established and optimized by changing the primer volume and annealing temperature. The specificity and sensitivity of the optimized multiplex PCR were also evaluated, and then the multiplex PCR was applied to the detection of three kinds of integron-related elements in 222 Vibrio strains of mariculture source. The results showed that the above primers could amplify three bands of 569 bp, 430 bp and 651 bp specifically. The optimized reaction conditions were as follows:the volume of primers 0.3 μL for int1, 0.6 μL for int_SXT, and 0.6 μL for ISCR1, and anneal temperature 50℃. Under the conditions, the detection minimum limit was 0.02 ng/μL of genome DNA. The results of detction for the above three integron-related elements in 222 Vibrio strains, using the optimized multiplex PCR, were the same as that of the control assays. In short, this study provides an effective multiplex PCR method for the simultaneous detection of int1, int_SXT and ISCR1,which could be applied to studies on integron-associated antimicrobial resistance.