Herein, gold nanoparticles (Au NPs) were synthesized by pulsed laser ablation (PLA) in a mixed-phase solvent of acetonitrile and water. The size of Au NPs and the number of graphitic carbon (GC) layers were controlled by varying the ratio of the solvent mixture. The surface-enhanced Raman scattering (SERS) of the Au NPs was investigated using 10-3 M 4-aminobenzenethiol and 10-4 M 4-nitrobenzenethiol as probe molecules. The SERS activity strongly depended on the nanogaps between particles owing to the formation of hot spots. In the present work, the nanogaps were controlled by changing the amount of GC layers. No GC layers were produced in water, resulting low SERS intensity. In contrast, Au NPs prepared in 30 vol% of acetonitrile showed significant SERS enhancement, which was attributed to the optimal size of the GC-coated NPs and a reasonable gap between them. The obtained results revealed that Au NPs produced by PLA in liquid could be applied in SERS-based microsensors.