A novel method of one-step preparation of dual-labeled gold nanoparticle bio-probes was established by the electrostatic adsorption and the covalent bonding of gold nanoparticles with antibodies and thiol-modified oligonucleotides, respectively. Characterization of probes, the coverage and activity of antibodies and oligonucleotides on probe surfaces were detected. The results indicated that the gold nanoparticles labeled with antibodies and oligonucleotides possess good bioactivity and the coverage of oligonucleotide and antibody on a dual-labeled gold nanoparticle bio-probe was (92 +/- 20) and (8 +/- 3), respectively. The preparative method is simple and stable. The dual-labeled gold nanoparticle bio-probes have an application value in detection of ultramicro protein.