The research process for electrochemical sensors often involves complicated fabrication/operation processes and an off-signaling mode, which significantly limit their reproducibility and practicality. Here, we present an aptamer-based, target-responsive DNA hydrogel electrochemical sensing platform (Apt-HE-Sensor) designed for one-step, and signal-on target detection, thereby eliminating the need for electrode modification, liquid transfer, and washing steps. By integration of an aptamer-embedded DNA hydrogel as a barrier layer on a bare electrode, target recognition occurs from the solid-liquid interface to the solution-gel phase. The binding of the target to the aptamer triggers decomposition of the hydrogel barrier layer, which accelerates the diffusion of electroactive probes to the electrode surface. When tetracycline is utilized as the model target, the sensing platform achieves a wide linear range (1-1500 μmol/L) with a detection limit of 0.58 μmol/L, while maintaining an excellent detection performance in actual water samples. Furthermore, by substituting the aptamer, the platform demonstrates significant discrimination of the corresponding target, showcasing a significant level of universality. This method offers a straightforward and efficient solution for the on-site real-time monitoring of small molecules and biomolecules in environmental and health applications.