Hund’s rule coupling (J) has attracted much attention recently for its role in the description of the novel quantum phases of multi-orbital materials. Depending on the orbital occupancy,Jcan lead to various intriguing phases. However, experimental confirmation of the orbital occupancy dependency has been difficult as controlling the orbital degrees of freedom normally accompanies chemical inhomogeneities. Here, we demonstrate a method to investigate the role of orbital occupancy inJrelated phenomena without inducing inhomogeneities. By growing SrRuO3monolayers on various substrates with symmetry-preserving interlayers, we gradually tune the crystal field splitting and thus the orbital degeneracy of the Rut2gorbitals. It effectively varies the orbital occupancies of two-dimensional (2D) ruthenates. Via in-situ angle-resolved photoemission spectroscopy, we observe a progressive metal-insulator transition (MIT). It is found that the MIT occurs with orbital differentiation: concurrent opening of a band insulating gap in thedxyband and a Mott gap in thedxz/yzbands. Our study provides an effective experimental method for investigation of orbital-selective phenomena in multi-orbital materials.