This study developed a novel electrochemical biosensor for single-step detection of proteins based on proximity-dependent surface hybridization assay. This strategy relied on simultaneous recognition of a target molecule by a pair of affinity probes, which was a prerequisite for efficiently promoting the ferrocene-labeled tail sequences of the proximate affinity probe pair to hybridize together with surface-tethered oligonucleotide, thus triggering the redox current of ferrocene at the electrode. The strategy, as a universal methodology for developing high-performance biosensors, was demonstrated using an aptamer probe to a homodimer protein PDGF-BB, and the fabricated aptasensor showed intrinsic high sensitivity, excellent resistance to nonspecific interferences, and ready reusability.