Here we report the 3D photo-cross-linking strategy based on the use of α-cyclodextrin as an alternative surface chemistry for the preparation of small molecule microarray (SMMs). Surface plasmon resonance imaging (SPRi) was used in combination with SMMs as a detection technology. Small molecules of diverse structure containing different functional groups can be covalently captured on a single slide and displayed in various orientations in a given spot. Several known small molecules-protein complexes were selected to validate this platform. Furthermore, a comparison between 2D PEG and 3D α-cyclodextrin photo-cross-linking surface was performed using fluorescence and SPRi techniques. Data produced from both type of surfaces strongly favors the better performance of 3D over 2D surface in terms of loading capacity, spot morphology, signal response, and kinetics values. We believe that this strategy will expand the utility of small molecule microarray for high throughput screening against a variety of target proteins.