Abstract: Analyzing fatty acid profiles in cetacean organs is crucial for evaluating the toxicological impacts of pollutants (e.g., heavy metals, persistent organic pollutants), yet a standardized, eco-friendly, and universally applicable method for fatty acid extraction from multiple tissues of marine cetaceans remains lacking. Lipids play essential roles in metabolism and cellular structure, serving as components of cell membranes, mediators of cell signaling, and energy storage molecules, which are indispensable for all organisms. Traditional approaches relying on chloroform/methanol systems have posed significant risks of environmental contamination and toxicity to operators, while their applicability across diverse tissues (e.g., liver, lung, spleen, kidney, muscle) remains unverfied. This study aims to develop a green and universal fatty acid extraction approach coupled with gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF MS) for analyzing fatty acids from multi-organ tissues (liver, lung, spleen, kidney, muscle) of cetaceans, thereby providing a reliable technical solution for fatty acid detection in stranded marine cetaceans. The research systematically compared extraction efficiencies of fatty acids with different organic solvent systems, including binary mixtures (chloroform/methanol, methyl tert-butyl ether (MTBE)/methanol, n-hexane/isopropanol) and a ternary mixture (MTBE/chloroform/methanol). The results demonstrated that the MTBE/methanol (5:1, V/V) system exhibited superior performance over the others, extracting the highest diversity of fatty acids while reducing environmental pollution risks and toxicity to operators compared to traditional chloroform-based methods. Under optimal extraction conditions, the concentration of derivatization reagents was also optimized. Notably, changes in reagent concentration showed minimal impact on extraction efficiency. Subsequent analysis revealed significant variations in fatty acid compositions among the five organs (liver, lung, spleen, kidney, muscle) of melon-headed whales (Peponocephala electra), which were attributed to distinct metabolic pathways, physiological functions, and lipid contents. For instance, the liver contains up to 60.09% of unsaturated fatty acids, such as arachidonic acid (C20:4ω-6) and DHA (C22:6ω-3), which may be related to its important detoxification function; the lungs have a saturated fatty acid content of 72.82%, as a large amount of straight-chain saturated fatty acids enhances their mechanical stability; the spleen and kidneys have very similar fatty acid profiles due to overlapping physiological functions; muscles are rich in 50.11% saturated fatty acids, such as palmitic acid (C16:0), which are used for energy storage. This study fills the technical gap in the analysis of fatty acids in cetacean multi-tissues, providing the first systematic characterization of fatty acid profiles in five key organs. The established green extraction method not only enables detailed investigations in lipid metabolism, but also paves the way for assessing pollutant bioaccumulation in marine cetaceans. Future research may extend this approach to other cetacean species or conduct long-term monitoring of the impacts of environmental contaminants, enhancing our understanding of the ecological health of marine mammals.