In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D-Ag) has been developed by means of self-assembly of monoclonal diphtheria antibody (D-Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D-Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol-gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen-antibody reaction in a phosphate buffer solution (pH 7.0). The immobilized D-Ab exhibited direct potentiometric response toward D-Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long-term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0 ร 10 ร3 to 1.2 mg โข mL ร1 with a detection limit of 1.1 ร 10 ร3 mg โข mL ร1 . Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2 mol โข L ร1 glycine-hydrochloric acid (Gly-HCl) buffer solution and 0.25 mol โข L ร1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis.