The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab′)2 and Fab′, with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab′ fragments. First, an optimisation of cleavage protocol to generate these F(ab′)2 and Fab′ fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab′ fragments on mixed SAMs leads to a >2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies.