In this paper, a facile large-scale preparation of surface-enhanced resonance Raman scattering (SERRS) substrates for the determination of Rhodamine B (RhB) based on silver nanowires (Ag NWs) has been developed. The morphology, structure, and properties of as-prepared Ag NWs are characterized using ultraviolet–visible (UV-Vis) spectroscopy, field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD), respectively. Ag NWs were assembled onto glass slides through a self-assembly method. Moreover, in our experiment, as-prepared Ag NWs@glass were used as a SERRS substrate to detect RhB at the excitation wavelength of 532 nm. Experimental conditions such as pH value and soaking time on SERRS performance were studied and optimized. Under the optimized conditions, the SERRS intensity at 1648 cm−1 exhibited a linear relationship with the concentration of RhB in the range of 1.0 × 10−9–1.0 × 10−5 mol L−1 and detection limit (signal-to-noise ratio [S/N] = 3) is as low as 0.3 nmol L−1. The corresponding correlation coefficient of the linear equation was 0.996. This method based on Ag NWs@glass for the detection of RhB in three kinds of condiment was investigated. The limits of detection (LODs) for RhB were 0.35 µg/g in chili powder, 0.14 µg/g in chili sauce, and 0.02 µg/g in Chinese prickly ash. The relative standard deviations (RSD) were between 2.18% and 4.56% (n = 3) and recoveries at three levels were in the range of 80.0–98.7% for different spiked food products. Moreover, the results showed that the proposed method was sensitive, convenient, and feasible for the determination of RhB in condiments.