This study used automatic control methods to prepare gold nanoparticles (AuNPs) as the substrate and rhodamine 6G molecule as the probe to investigate the enhancement effect, stability, and consistency of surface-enhanced Raman spectroscopy (SERS). The gold nanosols were prepared via automatic control using loop flow-reactor technology, and the synthesis of nanoparticles with different sizes was precisely controlled by optimizing the ratio of the solution required for the reaction between sodium citrate and chloroauric acid during the preparation process. The morphology, structure, and optical properties of the prepared AuNPs were investigated using field-emission scanning electron microscopy, transmission electron microscopy, and ultraviolet visible spectroscopy. Using the proposed method, AuNPs with average particle sizes of 72, 85, 93, and 103 nm were synthesized in a precisely controlled manner. The 93 nm particles exhibited good SERS activity for rhodamine 6G under 785 nm excitation with a detection limit of 2.5 × 10−10 M. The relative standard deviation of the SERS spectra synthesized multiple times was <3.5%, indicating their good sensitivity and reproducibility. The results showed that the AuNPs prepared by the automatic control of the loop-flow method have high sensitivity, stability, and reproducibility. Moreover, they exhibited notable potential for in situ measurement and quantitative analysis using SERS.