The synthesis of magnetite (Fe3O4) nanoparticles with the addition of polyethylene glycol-4000 (PEG-4000) was successfully carried out using a two-step co-precipitation method for the possible biosensor applications. In this study, different concentrations of PEG on Fe3O4/PEG (1:0, 4:1, 3:1, 2:1 and 1:1) were used. The crystallite size of the Fe3O4 decreased (14.35 to 12.83 nm) with the increase of PEG concentrations. The presence of a new carbonyl group in the samples indicates the presence of PEG in the Fe3O4. The weight loss percentage of the Fe3O4 increased from 7.7 to 41.46% with the increase of PEG concentrations. With the addition of PEG, Fe3O4 have a well-defined and homogeneous shape due to the presence of PEG which acts as a dispersing and stabilizing agent characterized. The magnetic properties showed that all samples exhibited superparamagnetic behavior. The saturation magnetization values of Fe3O4 decreased with increasing PEG layer and reached values of 65.34 emu/g with zero remanent magnetization and coercivity field while maintaining the behavior superparamagnetism and high saturation magnetization. To evaluate the magnetic properties of the samples to the sensitivity of biosensor, GMR-biosensor was used. Five types of Fe3O4/PEG-alcohol solutions with a concentration of 50 μg/mL were dropped on the surface of the sensor. It was found that there was a decrease in the |ΔV| value with the increase of PEG concentrations, undergoing a linear function |ΔV| = 97.31x+1880.97. Thus, the remarkable fact of saturation magnetization and controllable particle size in these Fe3O4/PEG can serve as promising biosensor applications.