Electrochemiluminescence (ECL) is the chemiluminescence triggered by electrochemical reactions. Due to the unique excitation mode and inherent low background, ECL has been a powerful analytical technique to be widely used in biosensing and imaging. As an emerging ECL luminophore, semiconductor quantum dots (QDs) have apparent advantages over traditional molecular luminophores in terms of luminescence efficiency and signal modulation ability. Therefore, the development of an efficient ECL system with QDs as luminophores is of great significance to improve the sensitivity and detection flux of ECL biosensors. In this review, we give a comprehensive summary of recent advances in ECL using semiconductor QDs as luminophores. The luminescence process and ECL mechanism of semiconductor QDs with various coreactants are discussed first. Specifically, the influence of surface defects on ECL performance of semiconductor QDs is emphasized and several typical ECL enhancement strategies are summarized. Then, the applications of semiconductor QDs in ECL biosensing are overviewed, including immunoassay, nucleic acid analysis and the detection of small molecules. Finally, the challenges and prospects of semiconductor QDs as ECL luminophores in biosensing are featured.