Abstract The binding of dodecyltrimethylammonium bromide (DTAB) with α-amylase was investigated under various experimental conditions, such as pH, ionic strength, urea and protein concentration at 25 °C using surfactant membrane-selective electrodes as a fast and accurate method. The obtained binding isotherms have been analyzed and interpreted using the Wyman binding potential concept. The results represent: a) the self aggregation of protein that occurs at enzyme concentrations of more than 1 mg/mL (this observation was also confirmed by light-scattering measurements), b) the binding affinity at 10−3 M NaBr is more than other salt concentrations, and c) in the concentration range of 3 to 5 M of urea a predominant unfolding of protein occurs.