To realize immobilization and hybridization of nucleotides, self-assembled monolayers (SAMs) containing anthryl groups which interact with nucleotides were prepared on a gold substrate. We used an unsymmetric disulfide containing an anthryl group (Anth-s-s-C8), which can form a SAM on a gold substrate, to immobilize nucleotides on the gold surface via the intercalation between DNA and anthryl groups. The SAM was characterized by time-of-flight mass spectroscopy. The interaction between polynucleotides and the SAM and hybridization were investigated by surface plasmon resonance. The surface coverage of anthryl groups on the SAM could be controlled by mixing Anth-s-s-C8 and 11-mercapto-1-undecanol in solution at various ratios. The efficient hybridization of polynucleotides could be realized by controlling 30% of Anth-s-s-C8 on the surface. This method can be applied to oligonucleotides, and the efficiency of hybridization is enhanced by introducing the double-stranded portion in the middle.