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A novel mushroom-like electrochemical immunoassay for the ultrasensitive detection of histone acetyltransferase activity (HAT p300) has been established on account of the new composite graphene oxide (GO) nanolayer. The immunoassay involves immobilization of substrate peptide onto Au electrode, acetylation in lysine of substrate peptide, and the interaction between acetyl group of lysine and acetyl-antibody (Ab Ac) of the GO nanolayer. The GO nanolayer comprises large amounts of methylene blue molecules (MB), giving rise to large signal amplification. Only in the presence of HAT p300, an obvious electrochemical signal appears and the peak linear current is proportion to the HAT p300 concentrations ranging from 0.01 to 150 nM with a detection limit of 0.0036 nM. The great enhancement on sensitivity of the proposed mushroom-like immunosensor derives from both the constructed Faraday cage and the extended outer Helmholtz plane (OHP). Further, the immunosensor with excellent sensitivity and selectivity can be applied for the HAT p300 activity detection in Hela cell lysates, serum and urine, hinting an improved and splendid analytical performance. Briefly, this stable, simple and ultrasensitive electrochemical immunoassay has considerable promise for further applications in the HATs-interrelated epigenetic studies and drug development. A novel mushroom-like electrochemical immunosensor for the ultrasensitive detection of histone acetyltransferase activity (HAT p300) has been reported, which shows promise for further applications in the HATs-targeted epigenetic research and drug discovery. Image 1 • An ultrasensitive mushroom-like electrochemical immunosensor is developed. • The proposed strategy can be applied for probing the HAT p300 activity. • The detection sensitivity of the novel method is improved sharply. • Mechanism is the constructed Faraday cage and extended outer Helmholtz plane. • The HAT-based immunosensor is also adopted for the screening of inhibitor. [ABSTRACT FROM AUTHOR]