DNA walkers, one of the artificial molecular machines which are constructed via smart synthetic DNA, have attracted rapidly growing attention from researchers in the biosensing field. In this work, we design an Exonuclease III (Exo III)-aided target-aptamer binding recycling (ETBR) activated bipedal DNA machine for highly sensitive electrochemical detection of antibiotics. To the best of our knowledge, this is the first time that a bipedal DNA machine has been applied in electrochemical sensing for antibiotics. On the one hand, the bipedal DNA walker exceeds the conventional single swing arm DNA walker in terms of walking efficiency and stability. On the other hand, the ETBR strategy, along with efficient strand displacement amplification via stepwise movement of a bipedal DNA walker significantly promotes the signal amplification efficiency. Under optimal conditions, this bipedal DNA machine possesses a detection limit of 7.1 fM within a linear detection range from 10 fM to 100 pM. Moreover, this electrochemical biosensor is expected to detect a wide variety of analytes using the corresponding target recognition probes. Thus, our proposed strategy offers a highly efficient, stable and practical platform for small molecule analysis.