Giant magnetoresistive (GMR) biosensors have emerged as powerful tools for ultrasensitive, multiplexed, real-time electrical readout, and rapid biological/chemical detection while combining with magnetic particles. Finding appropriate magnetic nanoparticles (MNPs) and its influences on the detection signal is a vital aspect to the GMR bio-sensing technology. Here, we report a GMR sensor based detection system capable of stable and convenient connection, and real-time measurement. Five different types of MNPs with sizes ranging from 10 to 100 nm were investigated for GMR biosensing. The experiments were accomplished with the aid of DNA hybridization and detection architecture on GMR sensor surface. We found that different MNPs markedly affected the final detection signal, depending on their characteristics of magnetic moment, size, and surface-based binding ability, etc. This work may provide a useful guidance in selecting or preparing MNPs to enhance the sensitivity of GMR biosensors, and eventually lead to a versatile and portable device for molecular diagnostics.