Immunosensor sensitivity is strongly dependent on the density of free active epitopes per surface area, which could be achieved via well-oriented immobilization of antibody fragments as bioreceptor molecules. Here, we report on the development of an electrochemical gliadin immunosensor based on the spontaneous self-assembly of antigliadin Fab fragments (CDC5-Fab) on Au surfaces. The analytical performance of this immunosensor is compared with a similar containing whole CDC5 antibodies previously modified with thiol groups (CDC5-SH) as the recognition element. Fab fragments were generated by reduction of the disulfide bond of F(ab)(2) fragments obtained by bromelain digestion of CDC5 antibody. Surface plasmon resonance (SPR) was used to evaluate the degree of immobilization and recognition ability of immobilized CDC5-Fab and CDC5-SH on gold surfaces. The studied surface chemistries were evaluated in terms of time required for SAM formation, stability, susceptibility to nonspecific interactions, and sensitivity using surface plasmon resonance, electrochemical impedance spectroscopy (EIS), and amperometry. CDC5-Fab formed a stable monolayer on gold after 15 min and retained >90% of antigen recognition ability after 2 months of storage at 4 degrees C. Detection of gliadin of Fab modified electrodes was evaluated by impedance and amperometry. Labeless impedimetric detection achieved a LOD of 0.42 microg/mL while the amperometric immunosensor based on Fab fragments showed a highly sensitive response with an LOD of 3.29 ng/mL. The Fab based immunosensor offers the advantages of being highly sensitive, easy, and rapid to prepare, with a low assay time.