The early and rapid diagnosis of acute myocardial infarction (AMI) is of great significance to its treatment. Here, we developed an electrochemiluminescence biosensor based on an entropy-driven strand displacement reaction (ETSD) and a tetrahedral DNA nanostructure (TDN) for the detection of the potential AMI biomarker microRNA-133a. In the presence of the target, numerous Ru(bpy)32+-labeled signal probes (SP) were released from the preformed three-strand complexes through the process of ETSD. The ETSD reaction cycle greatly amplified the input signal of the target. The released SP could be captured by the TDN-engineered biosensing interface to generate a strong ECL signal. The rigid structure of TDN could significantly improve the hybridization efficiency. With the assistant of double amplification of TDN and ETSD, the developed biosensor has a good linear response ranging from 1 fM to 1 nM for microRNA-133a, and the detection limit is 0.33 fM. Additionally, the constructed biosensor has excellent repeatability and selectivity, demonstrating that the biosensor possesses a great application prospect in clinical diagnosis.