The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.