Selectivity of electrocatalysts is determined not only by active sites for specific substrate interactions but also by the efficiency of electronic coupling mediated by intervening matrices. Here, we demonstrate the design of electron transport pathways to achieve catalytic specificity by interfacing redox-active methylene green (MG) and semiconducting graphdiyne (GDY), a 2D multilayered π-staked carbon nanosheet. Optical spectroscopy, electrochemistry, and computational simulation unravel the formation of MG dimers within the interlayer space of GDY nanosheets and the consequential tuning of activation overpotential and electron transfer rates. The electron-hopping pathway by self-exchange of MG dimers in neighboring sheets accelerates oxidation of dihydronicotinamide adenine dinucleotide at 7.06 × 10-2 cm·s-1, while the electron-tunneling pathway directly through GDY film decelerates oxidation of ascorbic acid at 6.60 × 10-5 cm·s-1, further endowing the MG-intercalated GDY nanosheets with high selectivity in mediated bioelectrocatalysis. This study extends the applicability of GDY in selective electrolysis and provides a universal strategy for modulating electrochemical properties of low-dimensional materials with laminar subnano/nanostructure.