The increasing application of gold nanoparticles (AuNPs) in biomedicine requires extensive investigation of surface modification and stabilization to maximize their advantages for the diversity of more challenging biological utilization. Herein, a thiol-mediated multifunctional phospholipid ligand was designed while disclosing a zwitterionic nature to AuNPs. The ligand was synthesized by attachment to two bidentate lipoic acid (LA) anchor groups and incorporation of a zwitterionic phosphatidylcho-line (PC) group, allowing for excellent hydrophilicity. As demonstrated through ultraviolet-visible spectroscopy, appropriate 7 nm diameter AuNPs modified with a 1,2-dilipoyl-sn-glycero-3-phosphorylcholine (di-LA-PC) compact ligand exhibited the best colloidal stability in a high NaCl concentration of up to 217 mM, different temperatures, and a wide range of pH values from 3 to 11 when compared to the traditional surfactants or thiol-contained amino acid surface modification cases. These AuNPs are also stable without specific interaction to positively/negatively charged proteins, possibly leading to prolonged blood circulation after in vivo administration. Moreover, much more resistance to ligand competition of dithiothreitol was found than other thiol-coated AuNPs, which further highlighted their affinity in an aqueous system. Biocompatibility of the zwitterionic ligand di-LA-PC-modified AuNPs was finally evaluated by hemolysis and cytotoxicity tests. Cumulatively, the remarkable stability and biocompatibility of AuNPs, multicoordinated with a di-LA-PC ligand, potentially motivated them as a practical alternative for surface tailoring in biotechnology.