Electrochemiluminescence (ECL) has several advantages, such as a near-zero background signal, high sensitivity, wide dynamic range, simplicity, and is widely used for sensing, imaging, and single cell analysis. ECL luminophores are the key factors in the performance of various applications. Among various luminophores, small organic luminophores exhibit many intriguing features including good biocompatibility, facile modification, well-defined molecular structure, and sustainable raw materials, making small organic luminophores attractive for the use in the ECL field. Although many great achievements have been made in the synthesis of new small organic luminophores, solving various challenges, and expanding new applications, there are almost no comprehensive reviews on small organic ECL luminophores. In this review, we briefly introduce the advantages and emission mechanisms of small organic ECL luminophores, summarize the main types, molecular characteristics, and ECL properties of most existing small organic ECL luminophores, and present the important applications and design principles in sensors, imaging, single cell analysis, sterilization, and other fields. Finally, the challenges and outlook of organic ECL luminophores to be popularized in biosensing applications are also discussed.