Mass spectrometry (MS) is a powerful analytical tool that can detect and identify multiple kinds of compounds such as biomolecules with high sensitivity. Recently, various nanoporous solid substrates have been developed for organic-matrix-free laser desorption/ionization (LDI) of analytes. In this study, periodic mesoporous organosilica (PMO) films are demonstrated for the first time to be promising as solid substrates for LDI mass spectrometry (LDI-MS). UV-absorbing PMO thin films having surface open pores with diameters of 20–30 nm are prepared using a triphenylamine derivative as a precursor and an amphiphilic block copolymer as a structure-directing template, followed by nanoscale surface etching. Peptide analytes introduced into the mesopores can be efficiently detected on the order of sub-picomole without the addition of an organic matrix. In addition, the PMO film shows an advantage of superior shot-to-shot reproducibility compared to a conventional matrix-assisted LDI (MALDI) method on a simple and easy way. PMO films with high tunability of framework composition and functionality may pave the way to new solid substrates for LDI-MS.