The preparation of nanofluids is very important to their thermophysical properties. Nanofluids with the same nanoparticles and base fluids can behave differently due to different nanofluid preparation methods. The agglomerate sizes in nanofluids can significantly impact the thermal conductivity and viscosity of nanofluids and lead to a different heat transfer performance. Ultrasonication is a common way to break up agglomerates and promote dispersion of nanoparticles into base fluids. However, research reports of sonication effects on nanofluid properties are limited in the open literature. In this work, sonication effects on thermal conductivity and viscosity of carbon nanotubes (0.5 wt%) in an ethylene glycol-based nanofluid are investigated. The corresponding effects on the agglomerate sizes and the carbon nanotube lengths are observed. It is found that with an increased sonication time/energy, the thermal conductivity of the nanofluids increases nonlinearly, with the maximum enhancement of 23% at sonication time of 1,355 min. However, the viscosity of nanofluids increases to the maximum at sonication time of 40 min, then decreases, finally approaching the viscosity of the pure base fluid at a sonication time of 1,355 min. It is also observed that the sonication process not only reduces the agglomerate sizes but also decreases the length of carbon nanotubes. Over the current experimental range, the reduction in agglomerate size is more significant than the reduction of the carbon nanotube length. Hence, the maximum thermal conductivity enhancement and minimum viscosity increase are obtained using a lengthy sonication, which may have implications on application.