Improving dispersion stability of nanofluids through ultrasonication has been shown to be effective. Determining specific conditions of ultrasonication for a certain nanofluid is necessary. For this purpose, nanofluids of varying nanoparticle concentrations were prepared and studied to find out a suitable and rather mono-dispersed concentration (i.e., 0.5 vol.%, determined through transmission electron microscopy (TEM) analyses). This study aims to report applicable ultrasonication conditions for the dispersion of Al2O3 nanoparticles within H2O through the two-step production method. The prepared samples were ultrasonicated via an ultrasonic horn for 1-5h at two different amplitudes (25% and 50%). The microstructure, particle size distribution (PSD), and zeta potentials were analyzed to investigate the dispersion characteristics. Better particle dispersion, smaller aggregate sizes, and higher zeta potentials were observed at 3 and 5h of ultrasonication duration for the 50% and 25% of sonicator power amplitudes, respectively.