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The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor’s electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor’s potential as a bioanalytical device for field applications and border phytosanitary inspections.