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The authors demonstrate here a method for three-dimensional patterning of proteins and other biological molecules. The method employs femtosecond-laser-induced three-photon polymerization, a technique which enables the construction of arbitrary two-and three-dimensional structures of submicron resolution. Biotin is subsequently attached to the three-dimensional ͑3D͒ structures via UV-activated cross-linking. The integrity of the photolytically immobilized biotin is confirmed by detecting the binding of fluorescently labeled avidin via fluorescence microscopy and via a surface acoustic sensor technique. In all, the technique opens the way for the fabrication of structures with a wide range of biomaterials as well as studying their dynamics within complex 3D structures.