A two-layer colloidal system is developed for the study of colloidal diffusion over a two-dimensional periodic energy landscape. The energy landscape is made from the bottom layer of colloidal spheres forming a honey-comb crystalline pattern above a glass substrate. The corrugated surface of the bottom colloidal crystal provides a gravitational potential field for the diffusing particles in the top layer. The obtained population probability histogram P(x, y) of the diffusing particles is used to fully characterize the energy landscape U(x, y) via the Boltzmann distribution. The dynamical properties of the diffusing particle, such as its escape time tR and diffusion coefficient D are simultaneously measured from the particle's trajectories. The long-time diffusion coefficients D is found to be in good agreement with the theory for all colloidal samples studied. The experiment demonstrates the applications of this newly constructed colloidal energy landscape.