Iron is one of the essential trace elements required for maintaining life, and it participates in several physiological activities. Comprehensive direct targeting of iron in tissue slices will be more conducive to exploring the physiological processes of iron metabolism disorders. In this study, we introduce a novel matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) methodology designed to map iron distribution comprehensively across tumor tissues and specific brain regions. We selected the reactive matrix based on iron chelating agents to promote the covalent charge labeling of oxygen and iron, which solved the problem of the low detection limit of metal elements when using MSI. Our research expands beyond iron mapping to include the simultaneous detection of molecules associated with energy metabolism in tumor tissues, lipid molecules related to myelin in demyelination models, and both neurotransmitter and lipid molecules in Parkinson's disease (PD) models. This multifaceted approach has unveiled the potential roles of iron in tumor progression and the pathogenesis of neurodegenerative diseases, providing novel insights into its functions during pathological processes.