The ability to pattern small molecules and proteins on artificial surfaces is of importance for the development of new tools including tissue engineering, cell-based drug screening, and cell-based sensors. We describe here a novel "caged" thiol-mediated strategy for the fabrication of planar Substrates patterned with biomolecules using photolithography. A thiol-bearing phosphoramidite (3-(2'-nitrobenzyl)thiopropyl (NBTP) phosphoramidite) was synthesized and coupled to a hydroxyl-terminated amorphous carbon substrate. A biocompatible oligo(ethylene glycol) spacer was used to resist nonspecific adsorption of protein and DNA and enhance flexibility of attached biomolecules. Thiol functionalities are revealed by UV irradiation of NBTP-modified surfaces. Both the surface coupling and photodeprotection were monitored by Polarization Modulation Fourier Transform Infrared Reflection Absorption Spectroscopy (PM-FTIRRAS) and X-ray Photoelectron Spectroscopy (XPS) measurements. The newly exposed thiols are chemically very active and react readily with a wide variety of groups. A series of molecules including biotin, DNA, and proteins were attached to the surfaces with retention of their biological activities, demonstrating the utility and generality of the approach.