In this paper, electrochemical behavior of a diphtheria immunosensor modified with nano-size conductor and semiconductor, as a mode of silica/gold/silver nanoparticles, has been investigated. Potentiometric immunosensor, cyclic voltammetry, and electrochemical impedance techniques were used to investigate the immobilization of diphtheria antibody on silica/gold/silver colloids. In the impedance spectroscopic study, an obvious difference of the electron transfer resistance between the silica-colloid modified electrode and the gold/silver-colloid modified electrodes was observed. The cyclic voltammogram tends to be more irreversible on silica colloids. Subsequently, using the silica colloid-modified immunosensor by potentiometry as a mode, it is found that the resulting immunosensor exhibited fast potentiometric response (<3min), high sensitivity and good reproducibility. Moreover, analytical results of several specimens obtained using the silica colloid-modified immunosensor are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.