Electrochemiluminescence (ECL) has emerged as a powerful analytical tool for the detection of biomarkers and the imaging of cellular functional molecules, owing to its low background, high sensitivity, and excellent spatiotemporal resolution. This review first summarizes representative classes of ECL luminophores, including organic small molecules, inorganic nanomaterials, and structurally programmable frameworks and polymers, along with their characteristic properties and recent applications. Subsequently, recent advances in ECL biosensing for in vitro detection of biomarkers such as proteins, nucleic acids, and small molecules are discussed, with particular attention to the evolution of target analytes and breakthroughs in achieving ultra-low detection limits. Next, this review focuses on the cutting-edge applications of ECL imaging at single-cell level. By integrating spatial confinement, label-free imaging, and in situ co-reactant generation with diverse signal-amplification strategies, ECL technology enables dynamic, minimally invasive, and high-resolution imaging of single-cell secretions, membrane proteins, and intracellular molecules, underscoring its potential for resolving functional heterogeneity at the single-cell level. Finally, the current challenges and future directions in ECL biosensing and imaging are outlined.