The protection and stabilization of metal nanoparticles with gemini surfactants can greatly improve their catalytic and reductive activities as well as stability, expanding their practical applicability. In this study, gold nanoparticles were prepared using three quaternary ammonium salt-based gemini surfactants with different spacer structures (2C12(Spacer)) as protective agents, and their structures and catalytic activities were investigated. The size of the 2C12(Spacer)-protected gold nanoparticles decreased as the ratio of [2C12(Spacer)] to [Au3+] ([2C12(Spacer)] : [Au3+]) increased from 1 : 1 to 4 : 1. Furthermore, the stability of the gold nanoparticles was affected by the spacer structure and surfactant concentration. The gold nanoparticles protected by 2C12(Spacer) with a diethylene chain and oxygen atom in the spacer were stable even at low surfactant concentrations because the gemini surfactants sufficiently covered the surface of the gold nanoparticles, and the aggregation between the nanoparticles was suppressed. In addition, the gold nanoparticles protected by 2C12(Spacer) with an oxygen atom in the spacer exhibited high catalytic activities for the reduction reaction of p-nitrophenol and 1,1-diphenyl-2-picrylhydrazyl radical scavenging reaction because of their small size. Thus, we elucidated the effect of spacer structure and surfactant concentration on the structure and catalytic activities of gold nanoparticles.