The immobilization of antibodies to sensor surfaces with a specific orientation is critical in biochemical sensor performance since this determines the primary properties of the biosensor such as sensitivity and selectivity. Covalent coupling is stable and provides a more permanent binding of biological materials to the substrate. However, these techniques may affect essential functional groups on the antibody, resulting in the loss of bioactivity. Another potential obstacle is steric interference between antibodies when closely bound to the substrate. To overcome the problem of losing bioactivity in covalent coupling, a potential approach is to employ a long chain spacer to immobilize the antibody indirectly while maintaining separation of the biomolecule from the substrate. In this study, Poly (ethylene glycol) (PEG) spacers were employed for tethering Escherichia Coli (E.Coli) K99 pilus antibody to silicon wafer surfaces for the purpose of increasing the flexibility of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a series of spacer lengths were used to covalently attach the antibodies to silicon surfaces. X-ray photoelectron spectroscopy (XPS) and Atomic Force Microscopy (AFM) 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 and the recognition process for bacteria-antibody was investigated by attaching E.Coli on the end of an AFM tip. Distribution of unbinding force and rupture distance from the force-distance curved obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the detected specific interaction up to 90%. J600 exhibited better flexibility in overcoming the steric hindrance experienced with direct immobilization than other spacer lengths. Moreover, binding efficiency, rupture distance and force distribution of bacteria-antibody pairs can be elucidated with AFM for measurement. Upon the inspecting on histogram of unbinding force and rupture distance, it shows an elemental quantum force of 0.32 nN with a periodicity of about 87 nm for the E.Coli K99 pilus-antibody pair.