A colorimetric assay for the ultrasensitive determination of thrombin was presented, in which the cationic polythiophene derivative was used as catalyst of the 3,3′,5,5′-tetramethylbenzidine (TMB)–H₂O₂ reaction and the thrombin-binding aptamer (TBA) was used as inducing polymer's different conformation elements. It was found the cationic polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-t-hiophene hydrochloride] (PMNT), can catalyze the oxidation reaction of TMB in the presence of H₂O₂ to produce a blue color solution. The catalytic activity of PMNT on the TMB–H₂O₂ reaction was closely relevant to the conformation of PMNT. The absorbance of TMB–H₂O₂ was distinctly increased in the presence of TBA. With the addition of thrombin, TBA interacted with thrombin to form a G-quadruplex structure. The conformational change weakened the catalytic activity of PMNT and resulted in a decrease in the absorbance. The colorimetric sensor could detect thrombin down to 4 pM with high selectivity against other interfering proteins. This work is not only of importance for a better understanding of the unique properties of cationic polythiophenes derivative but also have great potential for medical diagnostics and therapy for human health.