Photoelectrochemical (PEC) bioanalysis has recently become a perspective pathway for biomolecular sensing, presenting new insights for disease diagnosis and treatment. Herein, we present a new cathodic PEC platform for detecting Src kinase in a label-free, non-immobilized, and high-throughput mode. Specifically, treatment of the tyrosine-containing peptide by tyrosine kinase produced an ortho-diphenol structure of tyrosine that can in situ introduce oxygen vacancies (Ov) on the surface of NiTiO3 material, which led to the augmentation of the photoelectric signal. While the presence of the target Src kinase phosphorylated tyrosine in substrate peptide and blocked the surface Ov formation onto NiTiO3. Such a phenomenon was correlated with the Src kinase detection in terms of good sensitivity and selectivity, achieving the linear detection range of 0.001–2.0 μg/mL and the detection limit of 3.0×10−4 μg/mL (S/N=3). For Src kinase detection, this is a superior strategy to some classical methods in terms of easy operation, rapid response, and cost effectiveness. This study features the first exploration of cathodic photoelectrochemistry for Src kinase detection and is expected to expand its application in probing more disease related targets with unknown possibilities.