Imaging of lipids of whole-body specimens in two-dimensional (2D) analysis provides a global picture of the lipid changes in lipid-disturbed diseases, enabling a better understanding of lipid functions and lipid-modulation processes in different organs. However, 2D imaging of a single cross section can hardly characterize the whole-body lipid alterations. In this work, a three-dimensional matrix-assisted laser desorption/ionization mass spectrometry imaging (3D MALDI-MSI) approach was developed for analysis of whole-body zebrafish, for the first time, and applied to identify altered lipids and map their spatial distributions by using a zebrafish model of Niemann-Pick disease type C1 (NPC1), a neurovisceral lipid storage disorder causing both neurodegenerative disorder and visceral organ damage. The constructed 3D fish model provided comprehensive information on the 3D distribution of lipids of interest and allowed direct correlations between these lipids and organs of the fish. Obtained results revealed that several sphingolipids and phospholipids showed significant alterations and exhibited different localization patterns in various organs such as the brain, spinal cord, intestines, and liver-spleen region in the npc1 gene mutant fish compared to those of the wild type. The whole-body 3D MALDI-MSI approach revealed unique lipid signatures for different NPC1-affected organs, which might offer insights into the link between the impaired lipid storage and subsequent clinical symptoms, such as neurodegeneration and hepatosplenomegaly.