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. Display omitted