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Reactive oxygen species (ROS) play important roles in organisms and are closely related to various physiological and pathological processes. Due to the short lifetime and easy transformation of ROS, the determination of ROS content in biosystem has always been a challenging task. Chemiluminescence (CL) analysis has been widely used in the detection of ROS due to its advantages of high sensitivity, good selectivity and no background signal, among which nanomaterial-related CL probes are rapidly developing. In this review, the roles of nanomaterials in CL systems are summarized, mainly including their roles as catalysts, emitters, and carriers. The nanomaterial-based CL probes for biosensing and bioimaging of ROS developed in the past five years are reviewed. We expect that this review will provide guidance for the design and development of nanomaterial-based CL probes and facilitate the wider application of CL analysis in ROS sensing and imaging in biological systems.