The invention relates to a construction of an electrochemical luminescence sensor for detecting acetyltransferase activity. The construction comprises the following steps: firstly, polishing, cleaning and activating a gold electrode, assembling captured DNA on the surface of the electrode through action of gold-sulfur bond, and enclosing non-specific binding sites on the surface of the electrode with MCH; then adsorbing a polypeptide chains through electrostatic action, performing acetylation treatment on the polypeptide chains under the action of HATp300, so as to separate from the surface of the electrode; secondly, performing hybrid chain reaction on the electrode in a hybrid chain solution containing two hairpin DNA, and reducing a silver cluster; and finally, detecting electrochemical luminescence signal of the assembled electrochemical luminescence sensor in the solution. The ECL biosensor has remarkable advantages on the quantitative analysis of HAT p300 and the activity analysis on HAT p300 in complicated cell lysis buffering solution.