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Abstract A non-enzymatic and highly electrocatalytic H 2 O 2 biosensor was proposed by using a novel electrode composed of hemin-capped biomineralized gold nanoparticles (Hem@AuNPs), reduced graphene oxide (rGO) and chitosan (CS). Owing to the excellent electrocatalytic activity of Hem@AuNPs and great conductivity of rGO, this developed biosensor is capable of ultrasensitive measuring H 2 O 2 in real-time. In addition, the introduction of CS resulted in a film on the electrode, which can enhance the stability by protecting the biosensor from external disturbances. Taking advantages of the peroxidase-like activities of the nanohybrids, the developed electrode exhibited outstanding electrochemical performance toward H 2 O 2 with high selectivity, fast response, superior sensitivity and good stability. More importantly, the lower determination limit of 9.3 nM and wider linear ranges of 5 orders of magnitude enable this biosensor to detect H 2 O 2 releasing from living Hela cells accurately. This work provides tremendous potential for real-time monitoring the secretion of H 2 O 2 in living cells. Highlights • Introduction of biomineralized AuNPs and rGO increases the electrochemical performance of hemin greatly. • The biosensor Hem@AuNPs/rGO/CS/GCE exhibits marvelous electrocatalytic activity towards the non-enzymatic detection of H 2 O 2. • This biosensor can be used to the real-time monitoring of H 2 O 2 released from living cancer cells with satisfactory result. [ABSTRACT FROM AUTHOR]