The offshore waters of southern Zhejiang are rich in fishery resources and marine species; however, with the influences of overfishing, environmental pollution and other factors, fishery resources have been sharply reduced. Information on fatty acid content and composition can be used to evaluate the nutritive value of a species.In addition, because of the biological specifificity of fatty acids, essential fatty acids are transmitted stably between species and can track food sources and indicate trophic relationships among species. There are many studies analyzing the fatty acid contents of marine species, but there are few reports on the fatty acid contents of marine species specifically along the coast of Zhejiang Province, and few species have been involved. The purpose of this study was to provide references for consumers to choose seafood reasonably, as well as to provide basic data for the construction of food webs in the offshore waters of southern Zhejiang by exploring the characteristics of fatty acids in common marine species from these waters. This included an analysis of the nutritional value of fatty acids and exploring the relationship between fatty acid content and dietary habits. A total of 31 marine species were collected, including 20 marine fishes, 9 types of crustaceans, and 2 types of cephalopods. Three samples were randomly selected from each species for a total of 93 samples. Fatty acids were extracted using Folch’s method (chloroform-methanol solution, V : V = 2 : 1), were methyl esterified, and were then separated and detected using gas chromatography-mass spectrometry. Differences in the composition of the total lipids (TFA), saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-6 series of polyunsaturated fatty acids (n-6 PUFA), n-3 series of polyunsaturated fatty acids (n-3 PUFA), docosahexaenoic acid (C22:6n3, DHA), and eicosapentaenoic acid (C20:5n3, EPA) were analyzed. The results showed that the total lipid content of the marine fishes, crustaceans, and cephalopods ranged from 18.74–153.90 mg/g, 24.65–62.81 mg/g, and 37.23–92.18 mg/g respectively; the ranges of DHA+EPA content were 4.32–38.31 mg/g, 7.22–22.86 mg/g, and 12.48–49.61 mg/g, respectively. The contents of n-3 PUFA, EPA, and DHA in the 31 marine species from the offshore waters of southern Zhejiang were positively correlated with the content of TFA (P<0.01). There were significant differences among the 31 species of marine organisms in the offshore waters of southern Zhejiang. The average DHA/EPA ratios in marine fish and crustaceans in this study were 4.20 and 1.45, respectively. Additionally, the contents of DHA and EPA in crustaceans were relatively similar, whereas the content of DHA in marine fish was higher than that of EPA. DHA has been shown to be more effective than EPA at regulating blood pressure; thus, a higher intake of marine fish may help prevent cardiovascular disease. The mean ratios of SFA, MUFA, and PUFA in marine fish, crustaceans, and cephalopods (Uroteuthis duvauceli and Octopus variabilis) were 1.25 : 0.84 : 1, 1 : 0.67 : 1.75, 1 : 0.18 : 1.56, and 1 : 0.32 : 1.47, respectively; compared with the recommended ratio of 1 : 1 : 1, the content of MUFA was low. Therefore, consumers should consume a variety of seafood products to ensure a balanced intake of fatty acids. In addition, the results showed that the percentages of PUFA and n-3 PUFA were related to the dietary habits of the species, revealing that the species that mainly prey upon phytoplankton and benthic algae expressed higher contents than those of species that mainly prey upon swimming animals, such as fish.