Considering the central challenge of the simple and efficient strategy to generate sensitive analysis technology, herein, we proposed a novel electrochemiluminescence (ECL) strategy based on target-induced self-enrichment via hydrophobic interaction to generate significant ECL enhancement for untrasensitive detection of clinical biomarkers with cardiac troponin I (cTnI) for early diagnosis of acute myocardial infarction (AMI) as a model. Typically, the first antibody of cTnI (fAb) was immobilized onto the as-prepared electrode surface with the titanium dioxide nanoflower and gold nanoclusters When there was target cTnI, it could be captured onto the electrode surface based on the specific antigen-antibody interaction to furtherly capture cholesterol-modified second antibody of cTnI to increase the hydrophobicity of the electrode surface, which could be employed for the self-enrichment of hydrophobic ECL luminophore, tris(2,2'-bipyridyl-4,4'-dicarboxylato) ruthenium(II), and coreactant, tripropylamine in the detection solution. Thus, an increased ECL emission could be achieved due to the increased concentration of ECL luminophore and coreactant, which was quantitatively related with the concentration of target cTnI. As expected, a higher sensitivity was obtained with a detection limit of 0.04 pg/mL based on simplest operations of the proposed strategy with target-induced self-enrichment via hydrophobic interaction. Importantly, this hydrophobic interaction-based ECL strategy could be easily expanded to the bioassay of various biomarkers, providing an efficient tool for early clinical diagnosis of AMI and some other diseases.