To address the challenge of oil-water separation during the production of high-water-cut oil wells, this study focuses on mini-hydrocyclones, exploring their high-efficiency separation mechanism and the influence of structural parameters on separation performance. First, through theoretical analysis of the mini-hydrocyclone, it is found that it achieves efficient oil-water separation through the synergistic effect of a large radial resultant force, high pressure loss, and low Reynolds number. Subsequently, the Plackett-Burman (PB) experimental design method is employed to screen out the key structural parameters that significantly affect the separation efficiency, namely the overflow pipe diameter, large cone angle, and small cone angle. Based on the response surface methodology, these three parameters are optimized, and a mathematical model between the structural parameters and separation efficiency is established. The optimal structural parameters are determined as follows: overflow pipe diameter of 7.482 mm, large cone angle of 7.615°, and small cone angle of 2.810°. A prototype is fabricated using 3D printing technology, and indoor separation performance verification experiments are conducted. The results show that the relative errors between the simulated separation efficiency of the optimized mini-hydrocyclone and the experimental values, as well as the model prediction values, are 1.033% and 0.063%, respectively, indicating that the mathematical relationship model can effectively predict the separation efficiency of the mini-hydrocyclone within a certain range. In addition, simulation studies on separation performance are carried out under the working conditions of oil droplet sizes ranging from 20 to 300 μm and oil contents from 2% to 6%. The results demonstrate that the optimized mini-hydrocyclone exhibits excellent separation performance under the above conditions, providing theoretical and engineering basis for the efficient operation of downhole oil-water separation equipment.