Recently, there have been publications on preparing hybrid materials between noble metal and semiconductor for applications in surface-enhanced Raman scattering (SERS) substrates to detect some toxic organic dyes. However, the use of cuprous oxide/silver (Cu O/Ag) to measure the trace amounts of methyl orange (MO) has not been reported. Therefore, in this study, the trace level of MO in water solvent was determined using a SERS substrate based on Cu O microcubes combined with silver nanoparticles (Ag NPs). Herein, a series of Cu O/Ag ( 1-5) hybrids with various Ag amounts was synthesized via a solvothermal method followed by a reduction process, and their SERS performance was studied in detail. X-ray diffraction (XRD) and scanning electron microscopy results confirmed that 10 nm Ag NPs were well dispersed on 200-500 nm Cu O microcubes to form Cu O/Ag heterojunctions. Using the as-prepared Cu O and Cu O/Ag as MO probe, the Cu O/Ag5 nanocomposite showed the highest SERS activity of all samples with the limit of detection as low to 1 nM and the enhancement factor as high as 4 × 10 . The logarithm of the SERS peak intensity at 1389 cm increased linearly with the logarithm of the concentration of MO in the range from 1 nM to 0.1 mM.