Nanomaterial-derived quantum dots (QDs) are excellent electrochemiluminescence (ECL) luminophores and play an important role in optical sensing due to their excellent water solubility, good biocompatibility and tunable molecular size. In this work, a novel strategy was designed to form nano-hybrid Ti3C2 QDs-AuNPs in situ as a luminophore based on the unique reducibility of Ti3C2 QDs, which showed remarkable and stable ECL performance. Here, AuNPs were formed in situ without the addition of reducing agents and stabilizers, leading to threefold enhancement of the ECL signal of Ti3C2 QDs due to their excellent charge transfer capability. Meanwhile, Ti3C2 QDs-AuNPs with abundant Ti atoms also acted as recognition units. Through skillful combination with hybridization chain reaction (HCR) to expose more phosphate, an ECL platform was constructed to detect polynucleotide kinase (PNK) with good specificity and sensitivity. A lower limit of detection limit of 2.7×10−5 U mL−1 was achieved, with a wide linear relationship ranging from 0.0001 to 10 U mL−1. This novel strategy provides a guide for the application of nano-hybrid Ti3C2 QDs-AuNPs as a luminophore in the field of ECL bioanalysis.Graphical abstractNovel in situ-formed nano-hybrid Ti3C2 QDs-AuNPs were prepared as a luminophore, with threefold enhancement of the ECL signal of Ti3C2 QDs.