Recently, various semiconductor/metal composites have been developed to fabricate surface-enhanced Raman spectroscopy substrates. However, low metal loading on semiconductors is still a challenge. In this study, cystine was introduced to increase the accumulation of gold nanoparticles on zinc oxide, owing to the biomineralization property of cystine. Morphological analysis revealed that the obtained ZnO/Au/cystine composite not only had a higher metal loading but also formed a porous structure, which is beneficial for Raman performance. Compared with ZnO/Au, the ZnO/Au/cystine substrate displayed a 40-fold enhancement in the Raman signal and a lower limit of detection (10^–11 mol·L⁻¹) in the detection of rhodamine 6G. Moreover, the substrate has favorable homogeneity and stability. Finally, ZnO/Au/cystine displayed excellent performance toward crystal violet and methylene blue in a test based on river water samples. This study provided a promising method to fabricate sensitive semiconductor/noble metal-based surface-enhanced Raman spectroscopy substrates for Raman detection.