A simple and sensitive turn-off electrogenerated chemiluminescence (ECL) biosensor was designed for the analysis of histone acetyltransferases (HATs) activity and inhibitor evaluation based on the anti-hydrolysis ability of acetylated peptide and mimetic superoxide dismutase (SOD) features of tannic acid (TA) assembled gold nanoparticles (AuNPs) nanoprobes. In this strategy, after the acetylated reaction in the presence of HATs, the acetylated peptide on electrode was resistance to the hydrolysis of trypsin, and can absorb AuNPs@TA-Fe probe onto the electrode by the hydrophobic interaction and hydrogen bonding interaction. Thus, the ECL signal of the modified electrode in luminol solution decreased significantly owing to the mimetic SOD features of the TA assembled nanoprobe that can eliminate the reactive oxygen species. The ECL intensity changes can be utilized for sensitive HATs activities detection and inhibitor screening. The detection limit of the as-prepared ECL biosensor was 0.074 nM (S/N = 3). Moreover, the as designed ECL biosensor was also applied in MCF-7 cell lysates for HATs activity analysis and drug inhibition, which is feasible to HATs activity analysis and inhibitor screening, and presents highly promise in HAT-related clinical diagnostics.