Bifunctional catalysts comprising metal and acid sites are commonly used for many reactions. Interfacial acid sites impact intermediate reactions more than other sites. However, controlling the type and amounts of interfacial acid sites by regulating metal-support interaction (MSI) via traditional methods is difficult. Thus, the influence of MSI on interfacial acid sites remains unclear. We prepared Pt-mTiO 2 /α-Al 2 O 3 (m represents the cycle number of TiO 2 ) catalysts via atomic layer deposition (ALD). New Brønsted acid sites were generated via Pt-TiO 2 interaction, and the acidity was precisely regulated by regulating Pt-TiO 2 interaction by changing the TiO 2 nanolayer thickness. We chose levulinic acid (LA) hydrogenation as a model reaction. The catalytic activity varied with the TiO 2 nanolayer thickness and was linearly correlated with the Ti-OH species (Brønsted acid) content. Pt-40TiO 2 /α-Al 2 O 3, with the highest acid site content of 0.486 mmol/g, exhibited the best catalytic activity. Hydrogen spillover and water dissociation at the Pt-TiO 2 interface promoted Ti-OH species generation.