As one of the most abundant species of family Notothenioidei, rock cod (Patagonotothen ramsayi) plays an important role in energy transfer in the food web in the Patagonian Shelf. Currently, the work on feeding ecology of this species mainly bases on the conventional stomach content analysis, which only reflects short-term variation in feeding. Therefore, this study analyzed the distribution of fatty acids in three tissues (muscle, liver and gonad) of rock cod, and analyzed whether fatty acids in these tissues can characterize their feeding habits and transfer of diet. The results indicate that 27 kinds of fatty acids were detected in the tissues of rock cod. The storage and use of fatty acids in liver and gonad tissues are not suitable for indicating the diet of rock cod because they are involved in growth and reproduction. Compared to liver and gonad tissues, the longer renewal cycle of muscle tissue may better reflect its absorption of fatty acids from food. For the analysis of fatty acids in the muscle tissue of the rock cod in different size groups, it is inferred that the small-size rock cod (immature individual, 100−240 mm) mainly fed on planktonic organisms. The benthic feeding characteristics of rock cod increase with the increasing of size, and the feeding pattern transfer from planktonic feeding to planktonic-benthic feeding. In addition, due to the consumption of a certain amount of fishery discards by rock cod, the contribution rate of saprophytic food chain to predatory food chain is weakened. The results derive from this study further demonstrate that, compare to tissues of liver and gonad, the fatty acids of muscle tissue can be more suitable to indicate the food source of oceanic fish species.