Electrochemiluminescence (ECL) has been engineered to perform various tasks in the area of immunoassays and molecular diagnosis. However, there is still substantial potential for developments of ECL assay with high efficiency to achieve trace analysis. Herein, we demonstrate a polymer-amplified ECL assay via construction of linear Ru(bpy)32+-polymer. This new polymer material compensates for the relatively low ECL intensity from single ECL luminophore and realizes a stable and controllable labeling process. The polymer-amplified ECL assay achieved a remarkable sensitivity of 100 amol. The wide-ranging applications of the polymer-amplified ECL assay for Hepatitis B virus, carcinoembryonic antigen, 16sRNA, and thrombin also demonstrate its superiority. Hence, the polymer-amplified ECL assay possesses the potential to create a new paradigm in amplified ECL assays that could provide outstanding performance for biomedical analysis.