A separation-free electrogenerated chemiluminescence (ECL) immunoassay for biomarkers has been developed incorporating target assistant proximity hybridization and dynamically competitive hybridization of a DNA ECL signal probe for the first time. In this work, the biomarkers of acute myocardial infarction including cardiac troponin I (cTnI), cardiac troponin T (cTnT), and myoglobin (Myo) were chosen as the model proteins while the corresponding antibody was utilized as a recognition probe and the DNA5 tagged with ruthenium complex was chosen as an ECL signal probe (DNA5-Ru1). The biosensors were fabricated by covalently coupling the capture probe DNA1 onto the surface of a glassy carbon electrode, and then, a competitor ss-DNA2 was hybridized with DNA1. When the biosensor was incubated in the solution containing a target protein, the recognition probes (DNA3-Ab1 and DNA4-Ab2), DNA5-Ru1, and the coreactant tri-n-propylamine, the target protein was bounded with two antibodies of the recognition probes and thus induced the sufficient proximity hybridization of DNA3 with DNA1, DNA4 with DNA5-Ru1, and DNA5-Ru1 with DNA1 and the unwinding of the competitor DNA2 with DNA1, and ECL measurement was performed in separation-free format. It was found that the hybridization base number and length of DNA1 and a competing hybridization of DNA5-Ru1 with DNA2 for DNA1 have important effects. The developed ECL method showed a quite low detection limit of 0.4 pg/mL for cTnI, 0.5 pg/mL for cTnT, and 0.5 ng/mL for Myo. The fabricated biosensor exhibited stability and reusability. This work demonstrated that the developed ECL immunoassay is a promising separation-free and flexible strategy for quantitation of multiple proteins using one biosensor.