Enhanced efficiency for generating molecular ions is essential for high-throughput and sensitive detection using mass spectrometry in clinical diagnostics and biomarker discovery. In this study, we developed a novel strategy to promote laser desorption and ionization by using photonic crystals as substrates. The WO3-TiO2 inverse opal photonic crystal, with a coupling stop band and laser wavelength, significantly enhanced the efficiency of laser desorption and ionization owing to the slow light effect and the porous structure of the inverse opal, which increased the interaction between the laser and WO3-TiO2. Furthermore, stress biomarkers were conveniently measured under atmospheric pressure by using WO3-TiO2 inverse opal as an enhanced substrate to evaluate the impact of chronic unpredictable mild stress. The universal and highly sensitive substrate has promised for application in the highly sensitive detection and quantification of biomarkers.