The potential use of fatty acid (FA) profile as a dietary tracer in juvenile Chinese horseshoe crab Tachypleus tridentatus was assessed in a feeding experiment. Selected feed was provided to the juveniles at various growth stages according to their food sources in the wild: larval and adult brine shrimp Artemia salina for the first six months, followed by a mixture of short-neck clam Ruditapes philippinarum and greasyback shrimp Metapenaeus ensis for a further six months. Juvenile horseshoe crab haemolymph and tissues of book gill, opisthosomal spine, chelicera and swimming leg were sampled after the feeding experiment. The FA profiles revealed that the juvenile haemolymph and various tissues had a total of 23–24 FA components. The major FA components consisted of saturated (palmitic and stearic) at 22–34%, monosaturated (oleic) at 7–34% and polyunsaturated (eicosapentaenoic and arachidonic) FAs at 9–27%. According to principal component analysis, the FA signatures of juvenile haemolymph were greatly influenced by the shift of food sources from brine shrimp to clam and shrimp meat, whereas the FA profiles of different tissues were more resistant to the nutrient alteration. The present findings confirmed that the FA profile of both juvenile haemolymph and different tissues reflected the FA composition of recent and previous diets, respectively; and thus may be useful in indicating the foraging information of juvenile horseshoe crabs in the field.