A simple differential pulse voltammetric method based on a composite biosensor graphite/SiO2/Al2O3/Nb2O5/DNA (GRT/SiAlNb/DNA) was developed and validated for the determination of amitriptyline (AMT). The best results were obtained by using differential pulse voltammetry under measurements at pH 7.5 buffered with 0.20 mol L-1 phosphate buffer and DNA immobilization onto SiAlNb surface using 0.6 mg mL(-1) concentration. Validation parameters such as: selectivity, linearity, precision, accuracy, and stability presented results within the acceptable range. The sensitivity of sensor was found to be 0.216 mu A mu mol(-1) L, linear range from 10 to 80 mu mol L-1 and limit of detection of 0.12 mu mol L-1. Therefore, the developed method was successfully applied for the AMT determination in real samples of pharmaceutical formulations and can be used for routine quality control analysis of bulk pharmaceutical and pharmaceutical formulations containing AMT. (C) 2013 Elsevier B.V. All rights reserved.