Nanofluids are base fluids in which nanoscale particles are colloidal suspended. The research in the field of nanofluids becomes a hot topic due to their distinctive potential in applications involving heat transmission. The preparations and nanofluids’ stability are keen interesting topic for different applications of stable nanofluids. In this paper, CuO-DI water and CuO–ethylene glycol (EG) nanofluids were made without adding dispersant using two-step method, in which CuO nanoparticles (20 nm sized) with varying concentrations from 0.01, 0.1, 0.5, 0.8, and 1% by volume were used. The “magnetic stirrer with hot plate” at temperatures of 35 °C and 700 rpm was maintained for uniform distribution of nanoparticles contained in the base fluid and also does not allow the nanoparticles to aggregate. The nanofluid’s stability was ascertained using the sedimentation method. The synthesized CuO-based nanofluid sample was observed for the 5 days and the nanofluid’s stability was determined for the given sample. It was discovered that nanofluid concentrations greater than lower ones were less stable.