We report a novel method of one-step direct amination oil polycrystalline diamond to produce functionalized surfaces for DNA micropatterning by photolithography. Polycrystalline diamond was exposed to UV irradiation in ammonia gas to generate amine groups directly. After patterning, optical microscopy confirmed that micropatterns covered with an Au mask were regular in size and shape. The regions outside the micropatterns were passivated with fluorine termination by C3F8 plasma, and the chemical changes on the two different surfaces-the amine groups inside the patterned regions by one-step direct amination and fluorine termination outside the patterned regions-were characterized by spatially resolved X-ray photoelectron spectroscopy (XPS). The patterned areas terminated with active amine groups were then immobilized with probe DNA via a bifunctional molecule. The sequence specificity was conducted by hybridizing fluorescently labeled target DNA to both complementary and noncomplementary probe DNA attached inside the micropatterns. The fluorescence micropatterns observed by epifluorescence microscopy corresponded to those imaged by optical microscopy. DNA hybridization and denaturation experiments on a DNA-modified diamond show that the diamond surfaces reveal superior stability. The influence of a different amination time oil fluorescence intensity was compared. Different terminations as passivated layers were investigated, and as a result, fluorine termination points to the greatest signal-to-noise ratio.