We proposed an interface molecule for immobilization of DNA probes on solid substrates of DNA chips. We have designed and synthesized tripodal thiol derivatives for stable immobilization of oligonucleotide probes on a gold surface. On the basis of the tetrahedral structure of tripod, the tripodal thiol derivatives were bonded upright to the gold substrate, which would control the orientation of oligonucleotide probes. When the gold substrate with oligonucleotide probes tethered using the thiol derivatives was exposed to deionized water at higher temperatures, the tripodal interface molecules were attached to the gold surface more stably than the single contact molecules. The DNA chip platform combined with the functional interface molecule is suitable for a reproducible, inexpensive, and high-throughput detection system for genetic analyses in clinical diagnostics.