Impedimetric biosensors have received wide applications in bioanalytics for their sensitive detection and wide responding range. Donor-Acceptor-Donor (D-A-D) molecular architectures as signal transduction materials are emerging due to their efficient charge transportation. In this work, we synthesized a novel D-A-D molecule (BTCN) conjugated moiety. BTCN molecule can assemble onto glassy carbon electrode through strong π-π stacking, and immobilize tyrosinase (GCE@BTCN/Tyr/BSA), allowing the selective impedimetric detection of Dopamine, a key neurotransmitter that plays several essential roles in the brain and body. Remarkably, this biosensing system showed a linear range of detection from 1 nM to 500 μM, with a detection limit of 200 pM. This platform also demonstrated high biocompatibility and sensing specificity. We also find that the in-situ formation of copper-BTCN complex may contribute to the sensitivity and stability of the biosensing platform. We examined the DA level in the human serum sample of a healthy individual, demonstrating the great applicability of this electrochemical biosensing platform for practical use. © 2021 Elsevier B.V.