The aim of this research was to introduce a novel method to use zein protein and stabilize an ethanol-based emulsion. To study the effect of ethanol in the continuous phase of the emulsion on the stability and microstructure of the emulsions, ethanol content was tuned in the range of 50–90 %v/v. Also, the effect of zein concentration on the stability of ethanol-based emulsions was examined. The microstructure and mean droplet size of emulsions were measured using static light scattering (SLS), with Coherent Anti-Stokes Raman Scattering (CARS) Microscopy combined with image processing. The results showed that the effect of ethanol concentration on the interfacial tension and zein solubility plays a substantial role in the formation, stability, and microstructure of emulsions. The data indicated that 70 %v/v ethanol content resulted in the lowest interfacial tension between oil and ethanol-water mixture in the presence of 10 %w/v zein. Therefore, the minimum mean droplet size was achieved at 70 %v/v ethanol equal to around 10 μm. Furthermore, increasing zein concentration decreased oil droplet mean size and enhanced the emulsion stability against Ostwald ripening.