A stimuli-responsive chemiluminescent nanocomposite was developed based on bimetallic nanoclusters encapsulated in a reactive oxygen species (ROS)-responsive polymer for a highly sensitive determination of ROS (i.e., H2O2) in biological samples. Cobalt/gold bimetallic nanoclusters (GSH@Co-AuNCs) were synthesized using glutathione (GSH) as a reducing-cum-protecting reagent. The GSH-coated nanoclusters were covalently bound to N-(4-aminobutyl)-N-ethylisoluminol (ABEI) to form the GSH@Co-AuNCs-ABEI, which was further encapsulated in polymeric ROS-sensitive boronic ester modified dextran (Oxi-Dex) nanospheres through hydrophobic interactions. The stimulation of H2O2, as a model ROS, led to a sustainable structural cleavage of the Co-AuNCs-ABEI@Oxi-Dex nanocomposites and release of internal GSH@Co-AuNCs-ABEI, accompanied by intense chemiluminescence (CL). On this basis, an enzyme-free and reagent-free CL sensor was developed for a highly sensitive and selective determination of H2O2 with a detection limit of 35.8 pM in biological samples. It is of great application potential for the determination of ROS related to various diseases.