A novel photoelectrochemical biosensing platform for the detection of biomolecules at relatively low applied potentials was constructed using porphyrin-functionalized TiO2 nanoparticles. The functional TiO2 nanoparticles were prepared by dentate binding of TiO2 with sulfonic groups of water-soluble meso-tetrakis(4-sulfonatophenyl)porphyrin iron(III) monochloride (FeTPPS) and characterized by transmission electron microscopy; contact angle measurement; and Raman, X-ray photoelectron, and ultraviolet−visible absorption spectroscopies. The functional nanoparticles showed good dispersion in water and on indium tin oxide (ITO) surface. The resulting FeTPPS-TiO2-modified ITO electrode showed a photocurrent response at +0.2 V to a light excitation at 380 nm, which could be further sensitized through an oxidation process of biomolecules by the hole-injected FeTPPS. Using glutathione as a model, a methodology for sensitive photoelectrochemical biosensing at low potential was thus developed. Under optimal conditions, the proposed photoelectrochemical method could detect glutathione ranging from 0.05 to 2.4 mmol L−1 with a detection limit of 0.03 mmol L−1 at a signal-to-noise ratio of 3. The photoelectrochemical biosensor had an excellent specificity against anticancer drugs and could be successfully applied to the detection of reduced glutathione in gluthion injection, showing a promising application in photoelectrochemical biosensing.