Herein, an antibody-protein-aptamer electrochemical biosensor was designed by highly efficient proximity-induced DNA hybridization on a tetrahedral DNA nanostructure (TDN) for ultrasensitive detection of human insulin-like growth factor-1 (IGF-1). Impressively, the IGF-1 antibody immobilized on the top vertex of the TDN could effectively capture the target protein with less steric effect, and the ferrocene-labeled signal probe (SP) bound on the bottom vertex of the TDN was close to the electrode surface for generating a strong initial signal. In the presence of target protein IGF-1 and an aptamer strand, an antibody-protein-aptamer sandwich could be formed on the top vertex of TDN, which would trigger proximity-induced DNA hybridization to release the SP on the bottom vertex of TDN; therefore, the signal response would decrease dramatically, enhancing the sensitivity of the biosensor. As a result, the linear range of the proposed biosensor for target IGF-1 was 1 fM to 1 nM with the limit of detection down to 0.47 fM, which was much lower than that of the traditional TDN designs on electrochemical biosensors. Surprisingly, the use of this approach offered an innovative approach for the sensitive detection of biomarkers and illness diagnosis.