Preparation and hybridization of DNA-modified polycrystalline diamond substrates with fluorescently labeled complementary and noncomplementary oligonucleotide sequences were investigated. Hydrogen-terminated, free-standing diamond substrates were photochemically modified to produce amine-terminated surfaces. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used to characterize the initial attachment of a protected amine and the subsequent deprotection chemistry. Thiol-terminated DNA oligonucleotides were then linked to the amine-terminated diamond surfaces using a heterobifunctional linker. It is shown that hybridization on DNA-modified polycrystalline diamond is specific, with strong binding of perfectly matched 16-mer complements and little or no binding to 16-mers with 4 mismatched nucleotides. A direct comparison of DNA hybridization on DNA-modified diamond and DNA-modified surfaces of crystalline silicon shows that the diamond surfaces exhibit superior chemical stability under the conditions employed to hybridize and denature the DNA-modified surfaces.