In this paper, a novel graft copolymer, poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) with part of the PEG chains carrying a terminal nitrilotriacetic acid group (NTA) was synthesized. Through electrostatic interactions, these polycationic graft co-polymers assemble spontaneously from aqueous solution onto negatively charged surfaces, forming polymeric monolayers that present NTA groups at controlled surface densities on a highly PEGylated background. The NTA-functionalized PLL-g-PEG surfaces proved to be highly resistant to nonspecific adsorption in contact with human serum while allowing the specific and reversible surface binding of GFPuv-6His and β-lactamase-6His in native conformation. Micropatterns consisting of NTA-functionalized PLL-g-PEG in a background of PLL-g-PEG were produced using the "molecular assembly patterning by lift-off" technique. Exposure to Ni2+and GFPuv-6His resulted in a protein pattern of excellent contrast as judged by fluorescence microscopy. Furthermore, optical waveguide lightmode spectroscopy (OWLS) and a miniature fiber optic absorbance spectrometer (FOAS) were combined as affinity and catalytic biosensor to monitor in situ and quantitatively the amount of immobilized β-lactamase-6His and to determine the activity of the immobilized enzyme. The NTA-functionalized PLL-g-PEG surface is considered to be a promising sensor platform for binding 6 His-tagged proteins thanks to the simplicity and cost-effectiveness of the surface modification protocol, high specificity and nearly quantitative reversibility of the protein binding, and the potential to fabricate microarrays of multiple capture molecules.