Optimizing the atomic layer deposition (ALD) process of films is particularly important in preparing multilayer interference films. In this work, a series of Al2O3/TiO2 nano-laminates with a fixed growth cycle ratio of 1:10 were deposited on Si and fused quartz substrates at 300 °C by ALD. The optical properties, crystallization behavior, surface appearance and microstructures of those laminated layers were systematically investigated by spectroscopic ellipsometry, spectrophotometry, X-ray diffraction, atomic force microscope and transmission electron microscopy. By inserting Al2O3 interlayers into TiO2 layers, the crystallization of the TiO2 is reduced and the surface roughness becomes smaller. The TEM images show that excessively dense distribution of Al2O3 intercalation leads to the appearance of TiO2 nodules, which in turn leads to increased roughness. The Al2O3/TiO2 nano-laminate with a cycle ratio 40:400 has relatively small surface roughness. Additionally, oxygen-deficient defects exist at the interface of Al2O3 and TiO2, leading to evident absorption. Using O3 as an oxidant instead of H2O for depositing Al2O3 interlayers was verified to be effective in reducing absorption during broadband antireflective coating experiments.