Herein, we report a novel label-free electrochemiluminescent (ECL) biosensor for the detection of glycogen synthase kinase-3 beta (GSK-3β). A simple and feasible sensor was prepared by a two-step process. A polymeric coordination layer of phosphorylated poly vinyl with Zr 4+ was used as the sensory hosting matrix because it efficiently formed a complex. The exterior Zr 4+ can further combine with another phosphate through coordination, and GSK-3β catalyzes the phosphorylation of protein molecules. Thus, the biosensor can detect GSK-3β using luminol as an ECL probe. The ECL intensity of the proposed sensor responded proportionally to the concentration of GSK-3β under direct immersion mode with a linear response in a logarithmic scale over the wide range from 0.5 to 91.5 ng L −1 and a detection limit of 0.055 ng L −1 . Excellent selectivity, stability, and reproducibility were achieved using the prepared biosensor, which has a simple preparation, low cost, and disposable suitability. This work aims to provide a novel tool for early diagnosis and pathological mechanism exploration about AD by detecting inchoate change of GSK-3β content in body fluid, thus to precaution the risk of Alzheimer's disease. It is of great importance for clinical chemistry for the investigation of Alzheimer's disease.