We developed a novel protease assay device using nano-imprinted two-dimensional photonic crystal (2D-PhC) for the first time. 2D-PhC exhibits a characteristic reflection, and its intensity is strongly affected by small changes in refractive index (RI) of the 2D-PhC surface. In this study, protease substrate-immobilized 2D-PhC was newly developed in order to measure the protease activity, and the simple and selective detection of protease activity using 2D-PhC was successfully performed.