Surface-enhanced Raman spectroscopy represents a highly efficient detection method, offering significant advantages in the identification and detection of target analytes in food. To ensure that the substrate exhibits high sensitivity, it is essential to facilitate the formation of stable plasmonic "hot spot" regions. In this study, gold nanospheres (Au NSs) were synthesized on capillary substrates via in situ growth and seed-mediated method. The results demonstrated that optimal Raman signal enhancement was achieved when the APTES concentration was 20% and the diameter of the Au NSs was approximately 65 nm. The detection limits of fabricated substrate for Rhodamine 6G and crystal violet probe molecules are 10–11 and 10–10 M, respectively. In addition, aspartame molecules can be detected at levels as low as 0.0391 g/L, which is significantly below the national standard. The result demonstrates that the substrate has a high sensitivity, excellent uniformity, and high stability properties. Therefore, it is suitable for food additive detection with promising prospects for further development.