Electrochemiluminescence (ECL) is a light-emitting process triggered by the high energy redox between electrochemically oxidized and reduced luminophores or some coreactive intermediate radicals, representing a blooming hot topic over decades with a wide variety of bioanalytical applications. Due to the superb sensitivity, ultralow background noise, specificity, ease of integration, and real-time and in situ analysis, ECL has been developed as a convenient and versatile technique for immunodiagnostics, nucleic acid analysis, and bioimaging. Discovering highly-efficient ECL emitters has been a promising subject that will benefit the development of sensitive bioanalytical methods with prominent potential prospects. To date, the interdisciplinary integrations of electrochemistry, spectroscopy, and nanoscience have brought up the continuous emergences of novel nanomaterials which can be flexibly conjugated with specific bio-recognition elements as functional ECL emitters for bioassays. Therefore, a critical overview of recent advances in developing highly-efficient ECL emitters for ultrasensitive detection of protein biomarkers is presented in this review, where six kinds of the most promising ECL nanomaterials for biosensing and imaging of various disease-related protein biomarkers are separately introduced with references to representative works. Finally, this review discusses the ongoing opportunities and challenges of ECL emitters in developing advanced bioassays for single-molecule analysis and spatiotemporally resolved imaging of protein biomarkers with future perspectives.