Conventional solid/liquid electrochemical interfaces typically encounter challenges with impeded mass transport for poor electrochemical quantification due to the intricate pathways of reactants from the bulk solution. To address this issue, this work reports an innovative approach integrating a target-activated DNA framework nanomachine with electrochemically driven metal-organic framework (MOF) conversion for self-sacrificial biosensing. The presence of the target biomarker serotonin initiates the DNA framework nanomachine by an entropy-driven circuit to form a cross-linked nanostructure and subsequently release the Fe-MOF probe. Acting as a natural metal precursor and a nanoconfined source of reactant, the Fe-MOF probe is converted into electroactive Prussian Blue during electrochemical processes. Taking advantage of the confinement effect, our proposed biosensor exhibits the excellent capability to detect serotonin in a linear range from 1 pM to 5 μM with a remarkable detection limit of 0.4 pM and exceptional specificity against other interferents. The proof-of-concept demonstration of serotonin detection in clinical serum samples from patients with carcinoid tumors highlights the utility of a complex sample analysis. The design could be applied for other biomarker detection with a high potential to inspire innovative sensing approaches, holding promise for applications in biomedical research and disease diagnosis.