The effect of different concentrations of urea on the morphology of iron oxide (Fe3O4) magnetic nanoparticles was studied. Fe3O4 magnetic nanoparticles were fabricated by the coprecipitation method. The morphology, crystallinity, compositional purity, and emission characteristics were tested by the techniques of scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and Raman characterization. The drop-casting technique was successfully used to fabricate a potentiometric urea biosensor producing initially isopropanol and chitosan solution, consisting of Fe3O4 nanoparticles, on a glass fiber filter. To measure the developed biosensors voltage signal from the functionalized nanoparticles, a copper wire was utilized. The Fe3O4 nanoparticle surface functionalization was performed through the electrostatic immobilization of urease with the Fe3O4-chitosan (CH) nanobiocomposite. The presented urea biosensor measured a wide logarithmic range of urea concentration of 0.1-80 mM with a sensitivity of 42 mV/decade, and indicated a fast response time of approximately 12 s. The developed urea biosensor showed enhanced sensitivity, stability, reusability, and specificity. All experimental results demonstrate the application potential of the developed urea sensor for the monitoring of urea concentrations in human serum, drugs, and food industry-related samples.