A highly hydrophobic and non-toxic colloidal silica nanoparticle/polyvinyl butyral sol-gel composite membrane was prepared on a platinum wire electrode. With diphtheria-toxoid (D-Ag) as a model antigen and encapsulation of diphtheria antibody (D-Ab) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. It displayed a porous and homogeneous composite architecture without the aggregation of the immobilized protein molecules. The formation of immunoconjugate by a simple one-step immunoreaction between D-Ag in sample solution and the immobilized D-Ab introduced the change in the potential. Under optimal conditions, the D-Ag analyte could be determined in the linear ranges from 10 to 800 ng ml(-1) with a relatively low detection limit of 2.3 ng ml(-1) at 3 delta. The D-Ag immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy, and reproducibility. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications.