In the present investigation, ultrasonic studies of copper oxide (CuO) nanofluids are reported. Crystalline copper oxide nanoparticles are synthesized by co-precipitation method while the stable dispersions of nanosized CuO particles in ethylene glycol are prepared with the aid of sonication. The synthesized nanoparticles are characterized by X-ray powder diffractometry (XRD) to find the crystallinity and composition. Other characterization techniques such as SEM-EDX, TEM, and UV-visible are also provided to support the obtained results. Ultrasonic velocity, density and viscosity values are measured for different concentrations of CuO nanofluids at 25, 30 and 35oC. The acoustical parameters such as adiabatic compressibility (βad), intermolecular free length (Lf), relaxation time (τ), absorption coefficient (α/f2), acoustic impedance (Z), Gibb’s free energy (ΔG), free volume (Vf), rao’s constant (RM) and wada’s constant (W) are calculated from the experimental data. The inter particle interactions of nanoparticles and the cluster formation are realized through the variations in ultrasonic parameters. The results demonstrate that the aggregation of CuO nanoparticles becomes predominant at higher concentrations.