© 2016, Springer-Verlag Berlin Heidelberg.Acetylcholinesterase (AChE) biosensor based on conducting poly([2,2̍ʹ;5̍′ 2″]-terthiophene-3̍-carbaldehyde) (PTT) modified glassy carbon electrode (GCE) was constructed. AChE was immobilized on PTT film surface through the covalent bond between aldehyde and amino groups. The properties of PTT modified GCE were studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The biosensor showed an oxidation peak at +0.83 V related to the oxidation of thiocholine, hydrolysis product of acetylthiocholine iodide (ATCI), catalyzed by AChE. The optimum current response of the biosensor was observed at pH 7.5–8.0, 40 °C and 120 U/cm2 of AChE concentration. The biosensor showed a high sensitivity (183.19 μA/mM), a linear range from 0.015 to 1.644 mM, and a good reproducibility with 1.7 % of relative standard deviation (RSD). The biosensor showed a good stability. The interference of glycin, ascorbic acid, histidine, uric acid, dopamine, and arginine on the biosensor response was studied. An important analytical response from these inteferents that overlaps the biosensor response was not observed. The inhibition rate of malathion as a model pesticide was proportional to its concentrations from 9.99 to 99.01 nM. The detection limit was 4.08 nM.