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In this paper, a novel and sensitive electrochemical aptasensor for detecting tetracycline (TET) with prussian blue (PB) as the label-free signal was fabricated. A PB-chitosan-glutaraldehyde (PB-CS-GA) system acting as the signal indicator was developed to improve the sensitivity of the electrochemical aptasensor. Firstly, the PB-CS-GA was fixed onto the glass carbon electrode surface. Then, colloidal gold nanoparticles (AuNPs) were droped onto the electrode to immobilize the anti-TET aptamer for preparation of the aptasensor. The stepwise assembly process of the aptasensor was characterized by cyclic voltammetry (C-V) and scanning electron microscope (SEM). The target TET captured onto the electrode induced the current response of the electrode due to the non-conducting biomoleculars. Under the optimum operating conditions, the response of differential pulse voltammetry (DPV) was used for detecting the concentration of TET. The proposed aptasensor showed a high sensitivity and a wide linear range of 10?9 ～ 10?5 M and 10?5 ～ 10?2 M with the correlation coe?cients of 0.994 and 0.992, respectively. The detection limit was 3.2×10?10 M (RSD 4.12%). Due to its rapidity, sensitivity and low cost, the proposed aptasensor could be used as a pre-scanning method in TET determination for the analysis of livestock products.