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Objective Antimicrobial resistance (AMR) has become a global conc and is especially severe in China. To effectively and reliably Provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples. Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were Performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes. Results The new assay with limits of detection ranging from 40.9 to 8,000 copies／reaction. The sensitivity rate, specificity rate, Positive Predictive value, negative Predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with Positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. blaCTX-M, blaTEM, mcr-1, fexA, cfr, optrA, and intl1 showed over 80% Prevalence. The dissemination of AMR genes was distinct between the two farms. conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe Problem of AMR.