With the increasing rise of antibiotic-resistant pathogens, a simple and rapid detection of antibiotic resistance gene (ARG) is crucial to mitigate the spreading of antibiotic resistance. DNA-binding zinc finger proteins (ZFPs) can be engineered to recognize specific double-stranded (ds) DNA sequences in ARG. Here, we designed a simple and rapid method to detect ARG in bacteria utilizing engineered ZFPs and 2D nanosheet graphene oxide (GO) as a sensing platform. Our approach relies on the on and off effect of fluorescence signal in the presence and absence of target ARG, respectively. By taking advantage of the unique quenching capability of GO due to its electronic property, quantum dot (QD)-labeled ZFPs are adsorbed onto the GO sheets, and their fluorescence signal is quenched by proximal GO sheets through fluorescence resonance energy transfer (FRET). In the presence of target DNA, ZFP binding to the target DNA induces dissociation from GO, thereby restoring the fluorescence signal. Our system detects target DNA through restoration of QD emission as the restored signal increases directly with target DNA concentrations. Engineered ZFPs were able to detect specific dsDNA of the tetracycline resistance gene tetM with high specificity after only 10 min incubation on our GO-based sensing system. Our sensing system employed one-step FRET-based ZFP and GO combined technology to enable rapid and quantitative detection of ARG, providing a limit of detection as low as 1 nM. This study demonstrated the application of GO in conjunction with engineered DNA-binding domains for the direct detection of dsDNA with great potential as a rapid and reliable screening and detecton method against the growing threat of antibiotic resistant bacteria.