Zinc isotopic heterostructured zinc oxide thin films of 64ZnO/68ZnO/64ZnO were synthesized using pulsed laser deposition. The pulsed laser was first irradiated onto a polycrystalline target of 64ZnO to deposit the 64ZnO layer, then onto the 68ZnO target to prepare the 68ZnO layer and finally, the 64ZnO target was used again. The 64ZnO/68ZnO/64ZnO layered thin film was thus obtained. The thin films were annealed at various diffusion annealing temperatures. Diffusion profiles of the zinc isotopes due to the annealing were evaluated using secondary ion mass spectrometry (SIMS). The diffusion coefficients were slightly higher near the interface between the thin film and the substrate (the inner region) compared to the near surface (the outer region).