Fatty acids (FA) are a non-protein energy source and can act as trophic biomarkers in benthic food webs. We analysed the FA profiles of sea cucumber Apostichopus japonicus, comparing tissues of body wall, gut sediment and ovaries in two habitats. Rongcheng Bay: kelp raft cultivation area with high organic matter in sediment; Laoshan Bay: strong current with low sediment organic matter. The results showed that body wall and ovary tissues were rich in long chain polyunsaturated FA (LC-PUFA), which contributed ~31% to the FA dissimilarity between the two tissues. SIMPER (similarity percentages routine) results showed that C20:5ω3 (EPA), C18:1ω7, C20:4ω6 (AA), C16:0, C14:1 and C20:1ω11 contributed to dissimilarity between the body wall and ovary tissues, while 16:1ω7, 20:5ω3, C16:0, C18:1ω7, C18:0 and C14:1 contributed more to the dissimilarity of body wall tissues between the two habitats. FA biomarkers showed that sea cucumbers from the two habitats had different food sources, with brown kelp and vascular plants being the main food for sea cucumbers in Rongcheng and diatoms for those in Laoshan. To better understand differences in FA composition in sea cucumbers, more research is needed examining a wider diversity of tissue types and habitats.