The limited stability and random orientation of antibodies passively adsorbed onto solid supports are some of the main factors limiting the analytical performance of immune-based assay systems. Although the use of specific antibody-binding proteins led to significant enhancements in sensitivity, several uncertainties related to the orientation of these layers and the stability of the complexes formed with the antibodies need to be addressed for further progress. This paper introduces an alternative strategy based on the use of a charged polysaccharide layer for the stabilized and oriented assembly of antibody fragments. About one monolayer of F(ab′)₂ fragments of anti-human immunoglobulin G was passively adsorbed onto mercaptopropionic acid (MPA) and MPA/chitosan modified Au surfaces showing very good conformational stability. However, interrogation tests in the presence of human immunoglobulin G showed a piezoelectrical antibody binding signal about two times higher when the fragments were adsorbed onto chitosan. Given the similar coverage and conformational stability found in both cases and considering the different electrostatic scenarios, it is strongly suggested that the enhanced recognition of antigens may arise from the assembly of F(ab′)₂ mostly oriented in a hinge down end-on phase. Supporting this view, a limit of detection of about 3 μg mL⁻¹ was obtained from electrochemical methods. Although high, this is one of the best results reported (to the best of our knowledge) in proof-of-concept experiments using 2D electrically insulating immobilization layers with such a limited antibody loading capacity.