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Due to the limitations of conventional <i>Brucella</i> detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of <i>Brucella</i> in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of <i>Brucella</i> DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from <i>Brucella</i> genetic material. In addition, the detection specificity was examined using gDNA from <i>Brucella</i> and other zoonotic bacteria that may coexist in the same niche, confirming the method’s selectivity for <i>Brucella</i> DNA. Our proposed biosensor has the potential to be used for the early detection of <i>Brucella</i> bacteria in the field and can contribute to disease control measures.