In this study, a checkerboard arranged G4 nanostructure-supported electrochemical platform is developed well for the application to unique bio-enzymes examination. Herein, we focus on the two bio-enzymes involving histone acetyltransferase (HAT) and terminal deoxynucleotidyl transferase (TdT); the former leads to the acetyl transfer of acetyl coenzyme A to the lysine residue of the substrate peptide and the latter achieves the polymeric extension of DNA without template under a unique pool of dATP and dGTP (4: 6). A complex of antibody and short DNA is introduced onto the electrode surface based on the affinity interaction between acetyl in acetylated peptide and its antibody. and used for initiating reaction. Then, TdT-yielded rich-G sequence is formed to act as the backbone of checkerboard, and subsequently free G-DNAs can be stacked continually on the backbone under Mg2+. An excellent electrocatalytic signal to H2O2 emerges noticeably, which is related with the activity of HAT p300 and TdT with a low detection limit of 2.3 pM and 0.38 mU/mL, respectively. Finally, this strategy is also used successfully for the inhibitor screening and complex sample analysis, which has important implications for the advancement of HAT- and TdT-related electrochemical bioassay and drug discovery.