The abnormal level of neurotransmitter serotonin often leads to the blood-brain barrier. Thus, it is urgent to develop a sensitive and efficient method for serotonin detection. Here, we constructed a resonance energy transfer-based electrochemiluminescence (ECL-RET) biosensor sensitized with bipedal DNA walker for the sensitive determination of serotonin. Benefitting from the specific recognition of aptamer to target and enrichment ability of magnetic bead separation, this system achieved the signal transduction of serotonin to nucleic acid and excellent selectivity for serotonin analysis among various analogues. The spectral overlap between the emission spectrum of CdS QDs (energy donor) and the absorption of Ag nanoclusters (Ag NCs, energy acceptor) enabled effective ECL-RET. Through proximity ligation, bipedal DNA walker driven by catalytic hairpin assembly (CHA) significantly accelerated the reaction kinetics and resulted in the amplified responses. Relying on the ECL quenching, serotonin was quantified with a linear detection range of 1 pM-1 μM and a low detection limit of 0.28 pM. More notably, the practical application of the sensor was tested in human serum with recovery rates ranging from 95.0 % to 105.7 %. The combination of ECL-RET, proximity ligation and CHA-driven bipedal DNA walker enabled the biosensor to represent a step forward in neurological-related disease diagnosis.