Abstract <p>This study focuses on the production and characterization of titanium dioxide (TiO₂) nanoparticles (NPs) fabricated using an environmentally friendly technique, emphasizing their potential use in electrochemical biosensors. Knowledge of the physical and electrochemical properties of the NPs, such as their crystalline phases, shape, electrical conductivity, surface characteristics, and charge transfer resistance, is critical for enhancing their biosensor performance. Those properties were analyzed using ultraviolet-visible absorbance, X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The study also explored functionalization options for improving the interaction between TiO₂ surfaces and target analytes, increasing the overall efficiency of electrochemical sensors. The results showed that the synthesized particle size was 22 nm, indicating an increase in the surface area, allowing the glassy carbon/TiO₂-modified electrode to store input energy and then discharge, similar to a capacitor. Furthermore, the glassy carbon/TiO₂-modified electrode demonstrated high sensitivity and stability. The findings suggest that environmentally synthesized TiO₂ NPs can markedly accelerate the development of reliable and effective biosensing systems.</p>