We report laser desorption/ionization mass spectrometry using a dye-doped nanoporous polymer matrix. The nanoporous polymer matrix was fabricated through a holographic interference patterning technique. The periodically aligned nanopores in the resulting polymer matrix produced a high surface-to-volume ratio that facilitates the homogeneous cocrystallization of the matrix and an analyte (i.e., peptide in this demonstration). To generate nanostructures with further enhanced functionalities, dyes were also incorporated into the photopolymer. We demonstrate that by using the dye-doped nanoporous polymer matrix, we can identify peptides with an enhanced signal from the peptides and decreased noise from the ion fragmentation. These results indicate that the dye-doped nanoporous polymer matrix we use here can be a promising platform for laser desorption/ionization mass spectrometry.