Gold nanoparticles (GNPs) are attractive alternative optical probes and good biocompatible materials due to their special physical and chemical properties. However, GNPs have a tendency to aggregate particularly in the presence of high salts and certain biological molecules such as nucleic acids and proteins. How to improve the stability of GNPs and their bioconjugates in aqueous solution is a critical issue in bioapplications. In this study, we first synthesized 17 nm GNPs in aqueous solution and then modified them with six thiol compounds, including glutathione, mercaptopropionic acid (MPA), cysteine, cystamine, dihydrolipoic acid, and thiol-ending polyethylene glycol (PEG-SH), via a Au-S bond. We systematically investigated the effects of the thiol ligands, buffer pH, and salt concentrations of the solutions on the colloidal stability of GNPs using UV-vis absorption spectroscopy. We found that GNPs modified with PEG-SH were the most stable in aqueous solution compared to other thiol compounds. On the basis of the above results, we developed a simple and efficient approach for modification of GNPs using a mixture of PEG-SH and MPA as ligands. These biligand-modified GNPs were facilely conjugated to antibody using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide as linkage reagents. We conjugated GNPs to epidermal growth factor receptor antibodies and successfully used the antibody-GNP conjugates as targeting probes for imaging of cancer cells using the illumination of a dark field. Compared to current methods for modification and conjugation of GNPs, our method described here is simple, has a low cost, and has potential applications in bioassays and cancer diagnostics and studies.