The size of the TiB2 particles and the orientation relationship (OR) between the Al-TiB2 particles in Al solidified grains in Al-Cu hypo-eutectic alloy are characterized by a combined method of synchrotron multiscale-CT and SEM/EBSD to identify the true nucleation particle. Not only single TiB2 particles but also TiB2 agglomerates were observed in each of the four analyzed grains (A-D). The OR of {0001}TiB2∥{111}Al was confirmed for the largest TiB2 particle in each grain of A to C, corresponding to the free growth model. Such OR was absent in Grain D and EDS analysis revealed Cu segregation at the basal plane of its largest TiB2 particle. It is possible that the eutectic reaction of Al and Cu affects the nucleation and growth of Al, resulting in the change of OR. This paper evidently demonstrates that the eutectic reaction occurs on the nucleant TiB2 particles at the initial stage of nucleation.