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An argon-ion-induced carbon nanofiber (CNF) is expected to be an ideal nanocarbon for developing probes for scanning probe microscopes (SPM) owing to its superior physical properties and structures. To use this pristine CNF as a biofunctionalized SPM probe, its surface needs to be further modified with probing molecules that have specific biochemical affinities toward biological targets. In this study, using a carbon-coated Si wafer as a substitute for a CNF-tipped SPM probe, we investigated ion sputtering for the formation of an Au film on a carbon surface, followed by thermal annealing for making the Au-crystal orientation preferable for the chemisorption of thiolated biomolecules. Finally, we confirmed the biofunctionality of the immobilized thiolated oligonucleotide probe on the annealed Au film in order to hybridize it with the complementary oligonucleotide target. The hybrids were detected by fluorescence emitted from streptavidin-conjugated quantum dots tagged to the biotin molecules. From these results, it was shown that Au coating, thermal annealing, and the subsequent immobilization of thiolated biomolecules can be used for the development of new biofunctional CNF-tipped SPM probes.