Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins such as in serum samples without a separation step is of great significance to biomedical research but remains technically challenging. Major obstacles include nonspecific protein adsorption and quick fouling of the sensing interface. We take advantage of the unique transient stability of supported membranes and develop a new method using these membranes as a temporary and removable “cloaking” layer to eliminate the signals from non-specific interaction. A thin, hybrid self-assembled monolayer on gold was formed with 70 mol% mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles which fused to form a supported membrane. The protein analyte was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a non-ionic surfactant before the final measurement was carried out. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum was achieved with the membrane cloaking method, whereas direct measurement without membrane cloaking resulted in >300% error. Applications of the membrane cloaking strategy in developing highly sensitive, direct assay methods using SPR and enzyme-linked amplification mechanisms will be discussed.