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A properly designed sensing interface is crucial for the accurate and sensitive detection of biologically active molecules. Single-atom nanozymes from transition metal and nitrogen-doped carbon materials (M-N-C) have caught attention owing to their large surface area and strong bionic enzyme activity. Herein, a three-dimensional layered electrochemical electrode consisting of a Co–N–C nanoenzyme embedded in a reduced graphene oxide aerogel was prepared for the detection of hydrogen peroxide (H 2 O 2), dopamine (DA) and uric acid (UA). Due to its unique three-dimensional layered structure, rGA has excellent electrical conductivity and high material loading and is used to enhance the electrocatalytic performance of Co–N–C. The combination of single-atom nanozymes and electrochemical detection shows unique advantages in catalytic activity and selectivity. The limit of detection and detection range are 0.74 μM and 3–2991 μM respectively for H 2 O 2. Furthermore, it has been successfully implemented for the in-situ detection of H 2 O 2 in living cells. In addition, their simultaneous detection is also realized by the sensors for DA and UA. And it can accurately capture the signal of UA and DA in the urine. Meanwhile, the electrode displays satisfactory stability and repeatability. Therefore, this paper provides a new detection strategy for a variety of bioactive molecules, showing great potential in cell biology, pathophysiology and diagnostics. A novel electrochemical sensor based on single-atom nanozymes Co–N–C for bioactive molecules detection. [Display omitted] • Single-atom nanozymes Co–N–C exhibited peroxidase activity. • Co–N–C/rGA was used to detect H 2 O 2 in living cells, and successfully realized the simultaneous detection of DA and UA. • It provides a new method for the detection of bioactive molecules. [ABSTRACT FROM AUTHOR]