Compound-specific stable isotope analysis of individual amino acids (CSIA-AA) has been widely used in ecological and biogeochemical studies. It has been proven to be powerful in tracing the diet sources and trophic interactions. However, assessing the N sources of mesopelagic fishes has been inconclusive because the mesopelagic fishes\' unique domain (water depth ranged from 0 to 1000 m) and unresolved nitrogen isotopes of various forms. This study proposes a new method for coupling instruments (ion chromatography and Precon-IRMS) and chemical method of oxidation-reduction of amino acids, and also combined δ15N of AAs with δ13C of fatty acids (FAs) to analyze the trophic interactions of mesopelagic fishes in the South China Sea (SCS). AAs were isolated by ion chromatography with high peak resolution and collected by an automated fraction collector. The chemical method then converted the AAs into N2O with a robust oxidation yields and suitable molar ratio of NH2OH to NO-2. Finally, the δ15N of AAs at 20 nmol were measured with a reasonable precision (<0.6‰). With this method, this study report the first batch high precision δ15N of AAs and δ13C of FAs of mesopelagic fishes collected from SCS. Diaphus luetkeni, Chauliodus minimus and Bathygadus antrodes showed similar δ13C values of 20:4n-6 (～ ?28‰), while Argyropelecus affinis and Stomias had similar values (～ ?32‰). These results reflect that mesopelagic fishes had complex diet sources. An increase of 4‰ in δ15N of glutamic acid (Glu) was found between piscivorous and planktivorous fishes, which might suggest a trophic discrimination factor of mesopelagic fishes in the SCS. This study used δ13C of 20:4n-6 to reveal the diet sources of mesopelagic fishes and δ15N of Glu to clarify trophic level between piscivorous and planktivorous fishes. Thus, this combinative method could therefore ultimately be applied in a variety of deep-sea ecosystem.