Abstract: Deep-sea cold seep ecosystems are characterized by chemosynthesis-based trophic networks that support dense and diverse macrofaunal assemblages independent of sunlight. The trophic structure of six representative macrofaunal species—including Gigantidas haimaensis (mussel), Archivesica marissinica (clam), Paraescarpia echinospica (tubeworm), Branchipolynoe pettiboneae, Phymorhynchus buccinoides, and Neolithodes brodiei (king crab)—collected from the Haima cold seeps in the South China Sea, were investigated. Stable isotopes of carbon (δ¹³C), nitrogen (δ¹⁵N), and sulfur (δ³⁴S) of dissected soft tissues were analyzed, and the metabolic pathways and trophic networks were elucidated. Results show that G. haimaensis had extremely low δ¹³C values (63.9‰ to 52.5‰), indicating symbiosis with methane-oxidizing bacteria. In contrast, the clam and tubeworm exhibited higher δ¹³C values and markedly low δ³⁴S values, being consistent with sulfide oxidation. Predatory species such as B. pettiboneae that obtain energy from mussels by parasitic means, and P. buccinoides show intermediate δ¹³C and elevated δ¹⁵N values, suggesting dietary reliance on mussels. The king crab occupied the highest trophic level with the highest δ¹⁵N (8.8‰ to 11.2‰). The Bayesian mixing model (MixSIAR) was employed to estimate dietary contributions, allowing us to construct a trophic framework of the Haima seep ecosystem. This study highlights the application of multiple stable isotope tracers in resolving the carbon and sulfur cycling pathways and provided new insights into the biogeochemical interactions between cold seep fluids and benthic macrofauna.