This chapter focuses on the application of ECL imaging for quantitative detection of biomarkers and multiplexed assays. We will firstly introduce the basic concepts of ECL imaging biosensors and their structure. Secondly, we will discuss the application of ECL imaging immunosensors for biomarker detection, categorize and list biomarkers with respect to different diseases and their relevance, and explore the use of ECL immunosensors in biomarker detection, highlighting their broad application prospects in medical and non-medical fields. Subsequently, we will focus on the recent advances in multiplex ECL detection, including its combination with microfluidic chips, smart sensors, and multifunctional nanomaterials. Finally, this chapter will also analyze the challenges of ECL imaging in biomarker quantification and multiplexed assays in the context of specific application cases and consider the prospects for its future development, particularly in portable devices, high-throughput analyses, and for various fields such as precision medicine, environmental monitoring, and food safety.