Herein, an efficient photoelectrochemical (PEC) platform was constructed by a cosensitization strategy with a cascade energy level arrangement for the ultrasensitive evaluation of T4 polynucleotide kinase (T4 PNK). Based on CdSe quantum dots (QDs) with an extremely narrow bandgap, this cosensitization strategy offered a highly efficient sensitizer with a matching band-edge level of a ternary TiO2/g-C3N4/CdS nanocomposite. In this protocol, the ternary nanocomposite was first prepared to serve as the matrix to construct the PEC sensing platform. On the other hand, a well-designed hairpin DNA1 probe with 5'-hydroxyl termini was specifically phosphorylated by T4 PNK which would be selectively cleaved with lambda exonuclease (λ-Exo) outputting the 3'-thiol end ssDNA2. After tagged with CdSe QDs, ssDNA2 was captured by the complementary capture DNA3 on the electrode surface. As a result, CdSe QDs were in close contact with the ternary nanocomposite matrix, leading to an enhanced photocurrent response. Therefore, this proposed PEC platform displayed an analytical performance with a wide linear range from 0.0001 to 0.02 U mL-1 and a low detection limit down to 6.9 × 10-5 U mL-1. Moreover, this ternary nanocomposite-based platform exhibited excellent selectivity, good reproducibility, and remarkable storage stability, which shows great potential for T4 PNK detection and inhibitor screening.