A versatile biolinker for efficient antibody immobilization was prepared by site-specific coupling of protein G to DNA oligonucleotide. This protein G-DNA conjugate ensures the controlled immobilization of an antibody to the intended area on the surface of bioassay chips or particles, while maintaining the activity and orientation of the bound antibody. Streptococcus protein G tagged with a cysteine residue at the N-terminus was chemically linked to amine-modified, single-stranded DNA. SPR analysis indicated that the protein G-DNA conjugates sequence-specifically bind to complementary surface-bound DNA probes. More importantly, the resulting protein G, which is hybridized onto the DNA surface, possesses a greater antibody/antigen binding ability than even properly oriented protein G linked on the chip surface by chemical bonding. Antibody targeting on glass slides could also be achieved by using this linker system without modifying or spotting antibodies. Moreover, the protein G-DNA conjugate provided a simple but effective method to label DNA-functionalized gold nanoparticles with target antibodies. The DNA-linked protein G construct introduced in this study offers a useful strategy to manage antibody immobilization in many immunoassay systems.