A novel strategy was proposed here for fabrication of highly sensitive photoelectrochemical (PEC) sensor with prominent characteristics based on multifunctional photoactive materials and applied for ultrasensitive detection of glutathione (GSH) without additional catalysts. At first, growth of graphitic carbon nitride (g-C3N4) thin film on vertically aligned TiO2 nanotube arrays (TiO2-NTs) was achieved via facile thermal treatment of anodized Ti sheets over melamine which results in g-C3N4/TiO2-NTs heterostructure with well-controlled structure. In the next step, the electropolymerization was performed to load polythiophene (PTh) layer on the surface of g-C3N4/TiO2-NTs/Ti electrode. It was found that the combination of PTh with g-C3N4/TiO2-NTs nanocomposite exhibit excellent PEC response through an efficient electron transfer process under UV light irradiation and greatly improved the separation efficiency of photogenerated electrons and holes. With respect to our results, by injection of GSH, the analyte was oxidized by photogenerated holes during PEC reaction so that the photocurrent of the PEC sensor rose up. GSH could be quantified by measuring the photocurrent change. Under the optimized conditions, good linear relationship was obtained in the range of 1.0nM to 1.0μM with detection limit of 1.0×10−10 M (S/N=3). The proposed PEC sensing strategy exerted luminous analytical performance with high specificity, excellent stability and good reproducibility. Meanwhile, our as-prepared PEC sensor has been further explored for the determination of GSH in real sample, showing satisfactory results.