Based on ExoIII-assisted nucleic acid circulation and catalytic hairpin assembly (CHA) reaction, a dual-amplification electrochemical biosensors strategy for sensitive detection of tobramycin was presented. When the target was present, the trigger 1 (T1) was released to unfold hairpin DNA (H1), which allowed the ExoIII-assisted nucleic acid circulation to proceed smoothly and create a lot of trigger 2(T2). Following that, the CHA reaction between H2 and H3 proceeded under the catalysis of T2 and produced plenty of H2-H3 complex. Signal probe labeled with ferrocene was immobilized on the electrode ahead by adenine (PolyA). Finally, the H2-H3 complex combined with the probe and the DNA strand labeled with ferrocene (C2) fell off the electrode surface. The above process significantly reduced the electrochemical signal, so the biosensor has a low detection limit of 0.11 nM. Moreover, this biosensor has a satisfactory regenerability.