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A capacitive immunosensor on a glassy carbon electrode (GCE) was fabricated successfully by using an insulating poly (o-phenylenediamine) (OPD) film. Two methods, covalently coupling and copolymerization, were developed to immobilize the antitransferrin onto the GCE for sensitively probing transferrin. The capacitance change of the electrode caused by the interaction between antigen–antibody was monitored by the potentiostatic step. The immunosensors demonstrated a highly specific response to transferrin in a sample solution. The stable insulating property of the OPD film made immunosensor renewable in acid elution. For the immunosensor fabricated by the covalently coupling method, the linear detection range was 0.1 to 45.0 ng/mL, with a detection limit of 0.061 ng/mL (S/N = 3). For immunosensor fabricated by the copolymerization method, the linear detection range was 5.0 to 17.5 ng/mL, with a detection limit of 2.0 ng/mL. This indicated that the immunosensor prepared by the covalently coupling method showed better sensitivity and a wider detection range than one fabricated by the copolymerization method. Compared with self-assembled monolayers, the method of using the insulating OPD film simplified the process of immunosensor fabrication and provided a new approach to prepare capacitive immunosensor on carbon substrate. [ABSTRACT FROM AUTHOR]