Stable isotope analysis has become increasingly popular in the determination of trophic relationships in marine ecosystem. In this study, based on bottom trawl surveys, 33 species of fish and 18 species of invertebrates were collected in February and May 2016 in the offshore waters of southern Zhejiang. All of these species accounted for 90% of the total catch biomass, highlighting them as being the dominant fish species and invertebrates in the offshore waters of southern Zhejiang. Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) of the fishery organisms were calculated with an element analysis-isotope ratio mass spectrometer (EA-IRMS), and the δ¹⁵N values were applied to estimate their trophic levels. The results revealed that the major fishery organisms had great variations in their composition of stable carbon and nitrogen isotopes, especially the fish species, with statistical difference (C values of the fishery organisms ranged from -19.71‰ to -14.01‰, with an average of -17.06‰, and the δ¹⁵N values ranged from 7.05‰ to 13.69‰, with the average of 10.98‰. Filter-feeding bivalves were set as the baseline. The average trophic levels of fish, crustaceans, cephalopods and gastropods were 2.66-4.21, 3.08-3.72, 2.83-3.49 and 3.54-3.62, respectively. The major fishery organisms were mainly in the range of 3.0 to 4.0, most of which were primary and intermediate carnivorous species. Cluster analysis on the stable isotope results of the major fishery organisms produced organism groups that could be interpreted with trophic and feeding information. Based on hierarchical cluster analysis and feeding literatures, five feeding groups of the major fishery organisms from the offshore waters of southern Zhejiang were classified:zooplankton feeding, omnivorous, benthic feeding, mixed feeding and nekton feeding. The trophic structure of the food web could be divided into four trophic groups, which includes primary consumers, secondary consumers, intermediate consumers and senior consumers. According to the characteristics of trophic structure, the primary consumers were composed of omnivorous species, and the secondary consumers mainly included small fish, shrimps and cephalopods. The intermediate consumers were mainly benthic crabs, gastropods and mixed carnivorous fish, while the senior consumers were carnivorous fish. In this study, the continuous trophic spectrum of fishery organisms in the offshore waters of southern Zhejiang was established to provide an important reference for the study of energy flow and matter cycles in marine food webs.