In this work, a biosensor that uses gelatin and alginate hydrogels in addition to titanium dioxide (TiO2) nanoparticles (NPs) as a sensor matrix was developed to detect superoxide radicals (O2 •-), which play a role in carcinogenesis when present in excess levels. Parameters affecting the performance of the biosensor such as the amount of gelatin-alginate ratio, amount of TiO 2 NPs, the concentration of SOD enzymes, and glutaraldehyde (GA) cross-linker were investigated. Chronoamperometry was used as an electrochemical technique for the development of biosensor as well as characterization steps. The developed biosensor exhibited two linear ranges between 0.0009 mM -0.125 mM and 0.25 mM -2 mM, which was utilized as calibration curves. The detection limit of the biosensor was found 0.9 μM, which was at the appropriate level for the detection of O2 •-in tumor samples. Finally, the constructed biosensor showed significant analytical performance, such as low detection limit, reusability, and reproducibility.