Surface modification for biosensors has focused attention for improvement of their sensitivity and specificity, particularly for the detection in complex medium. In this work, we have synthesized zwitterionic carboxybetaine-thiols (CB-thiols) and sulfobetaine-thiols (SB-thiols) for modification of gold substrates to form a functional self-assembled monolayer (SAM) for the immunoassay in a surface plasmon resonance (SPR) biosensor. X-ray photoelectron spectroscopy (XPS), contact angle goniometer, and cyclic voltammetry were applied for characterizations of elemental composition, surface wettability, and packing density, respectively. The antifouling properties of the SAMs were accessed by quantitative analysis of protein and bacterial adsorption. The results from the SAMs with a single component indicated that the SB-thiol SAM provides better surface hydrophilicity, fouling resistance, and packing density as compared to the CB-thiol SAM, likely due to the ionic association of CB moieties. However, the CB-thiol with the functional carboxylate group plays a critical role in postmodification of biomolecules via commercially available amine coupling chemistry. Thus, the mixed SAMs were prepared to integrate the unique characteristics from CB- and SB-thiols to control compositions and surface properties. The immunoassay was performed in the SPR biosensor, showing that the zwitterionic mixed SAM enables immobilization of biorecognition elements (BREs), and improved sensitivity and specificity. Consequently, the work reveals excellent and attractive versatility, antifouling, and functionalizable properties of zwitterionic mixed SAMs comprising CB- and SB-thiols for biosensing applications. This surface chemistry is expected to be applicable to monitor specific molecular recognition events.