Reactive oxygen species (ROS), produced during oxygen metabolism, participate in and regulate various life processes. It is of great significance to monitor ROS in biological organs to further study oxygen metabolism. Herein, an ultrasensitive sensing platform is developed with electrochemiluminescent (ECL) signalling by integrating bioactive magnetic beads (BMBs) on indium tin oxide (ITO) coated glass using a magnet. For the first time, AuNPs were successfully deposited on Fe3O4 NPs in situ by reduction of α-ketoglutaric acid (α-KG), therefore the electroactive protein, haemoglobin (Hb) or cytochrome C (Cyt C), was assembled on via covalent bonds. The protein can realize direct electron transfer (DET) and catalyse the redox of ROS, reaching a detection limit of 6.21 μM or 0.6 μM of H2O2. Also Au@Fe3O4 NPs efficiently enhanced the ECL of luminol, promoting the sensing ability for ROS. This simultaneous effect endows the platform with low LOD of ROS for 7.69 nM (Hb), or 1.97 nM (Cyt C). Finally, the feasibility and practicality of the sensing platform were verified by monitoring the ROS released from mouse myocardial tissue.