Gold nanoparticles (AuNPs) have been extensively utilized in various fields such as sensors, life sciences, and catalysis. In this study, AuNPs were synthesized using a reduction method and subsequently treated with thiourea in an ethanol-water environment to prepare AuNPs film using a centrifugal deposition method for first time, resulting in the aggregation of the initial small-sized AuNPs into larger microsphere-like structures. The addition of thiourea facilitated the interconnection between AuNPs, ultimately leading to the formation of large stable gold microspheres. The sheet resistance of the AuNP films transitioned from being non-conductive to exhibiting a sheet resistance of 42.6 Ω/sq following thiourea treatment. The transformation from a flat surface to tightly connected particles resembling microspheres was observed from SEM images. The thiourea treatment not only altered the morphological characteristic of the AuNPs films but also significantly increased the number of scattering sites on their surface, leading to a substantial enhancement in the Raman scattering effect for methylene blue. This structural configuration also improved the electronic conduction and stability of the treated AuNPs films. Consequently, these findings suggest that AuNPs have promising application prospects in surface-enhanced Raman scatting (SERS), as well as in flexible electronics, catalysis, adsorption, and energy fields.