Dodecabenzylbambus[6]uril (Bn12BU[6]) is an anion receptor that binds the perchlorate ion the most tightly (stability constant ~1010 M−1) of all anions due to the excellent match between the ion size in relation to the receptor cavity. This new bambusuril compound was used as an ionophore in the ion-selective membrane (ISM) to develop ion selective electrodes (ISEs) for determination of perchlorate concentration utilizing the poly(3,4-ethylenedioxythiophene) (PEDOT) polymer film as a solid-contact material. Variation of the content of Bn12BU[6] and tridodecylmethylammonium chloride (TDMACl) in the plasticized poly(vinyl chloride)-based ISM was also tested. All the prepared solid-contact ISEs and their analytical performance were characterized by potentiometry, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronopotentiometry. The ISEs showed rapid response and a sub-Nernstian slope (~57 mV/decade) during potentiometric measurements in perchlorate solutions in the concentration range from 10−1 to 10−6 M simultaneously with their high stability and sufficient selectivity to other common inorganic anions like bromide, chloride, nitrate and sulphate. The function of the ISE was further verified by analysis of real water samples (lake, sea, and mineral water), which gave accurate and precise results.