Semiconductors have great potential as surface-enhanced Raman scattering (SERS) substrates due to their excellent physiochemical properties. However, they provide low signal enhancements relative to their plasmonic counterparts, which necessitates innovation in their synthesis and application. Substitutional atomic doping is proposed to improve SERS enhancement by controlling electronic properties, such as the band gap. In this work, zinc oxide (ZnO) nanoparticles were synthesized by co-precipitation and doped with magnesium (Mg) at concentrations ranging from 2–10%. Nanoparticle morphology and size were obtained by scanning electron microscopy (SEM). Elemental composition and chemical states were determined using X-ray photoelectron spectroscopy (XPS). Optical properties were obtained with a UV-vis spectrophotometer, while a Raman spectrometer was used to acquire Raman signal enhancements. Stability was assessed by UV-vis spectroscopy, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that the absorption edge of Mg-doped ZnO nanoparticles was red-shifted compared to pure ZnO nanoparticles. The band gap decreased (3.3–3.01 eV) with increasing Mg doping, while the highest Raman enhancement was observed at 2% doping. No significant cytotoxic effects were observed at low concentrations (3–12 μg/mL). Overall, this study provides evidence for the tunability of ZnO substrates and may serve as a platform for applications in molecular biosensing.