<p>The study aimed to develop a novel multiplex fluorescent PCR assay for the simultaneous detection of seven aminoglycoside resistance genes, which is crucial for optimizing aminoglycoside dosing regimens and addressing the growing issue of antibiotic resistance. Primers for the seven target genes were designed by Primer Premier 5.0. The multiplex fluorescent PCR was optimized by varying primer concentrations and annealing temperatures. The assay’s sensitivity was evaluated using purified recombinant plasmids containing the target genes. Ninety-nine clinical isolated strains were analyzed by the newly developed multiplex fluorescent PCR assay and reported traditional PCR assay in parallel. The multiplex fluorescent PCR successfully detected the seven aminoglycoside resistance genes with detection limits ranging from 47 to 189 copies per reaction (concentration giving a positive result in 95% cases). Analysis of clinical isolates showed the positive rates of these resistance genes ranged from 1.01 to 30.30% in clinical isolates. The genes <i>aac(6’)-I</i> and <i>ant(3’’)-I</i> exhibit relatively high positive rates. In contrast, the gene <i>aphA(3’)-VI</i> demonstrated a considerably lower positive rate. The detection results of our multiplex fluorescent PCR and a reported traditional PCR assay showed a high degree of consistency. This study successfully developed a multiplex fluorescent PCR assay that is simple, specific, sensitive, and capable of high-throughput detection of seven aminoglycoside resistance genes. This new tool shows promise for use in microbiology laboratories for rapid identification of aminoglycoside resistance genes.</p>