In this study, varieties of lipid bilayer-protected gold nanoparticles (AuNPs) were synthesized through a simple wet chemical method, and then the effect of freeze-thawing on the as-prepared AuNPs was investigated. The freeze-thawing process induced fusion or fission of lipid bilayers tethered on the AuNPs. The UV-vis spectra and transmission electron microscopy experiments revealed that the disruption of lipid bilayer structures on the nanoparticles led to the fusion or aggregation of AuNPs. The role of freeze-thawing in the evolution of lipid bilayer-protected AuNPs was studied. The addition of adequate sucrose, a well-known cryoprotectant, effectively prevented the fusion or aggregation of lipid bilayer-protected AuNPs undergoing the freeze-thawing process. The possible mechanism of sucrose preserving the integrity of the lipid bilayer-protected AuNPs was also discussed.