Arapaima gigas is an ancient, air-breathing, giant fish of Amazonian rivers. Due to its economic importance, the development of aquaculture for this species is currently a good alternative to counteract the decline of wild populations. Reproduction in captivity relies on the constitution of male and female pairs, which occur naturally in large ponds. But, as sex determination of A. gigas is not possible by morphological criteria it is very difficult to optimize reproduction success and fry production in each pond. This situation prompted us to develop a sexing methodology based on the detection of female specific plasma Vitellogenin (Vtg) using an Enzyme Immuno-Assay (EIA). Vitellogenin purification was performed by electro-elution after polyacrilamide gel electrophoresis (PAGE) from plasma of 17β-Estradiol treated A. gigas juveniles. Two different Vtg molecules were isolated, (Vtg1 and Vtg2) with 184 and 112 kDa apparent molecular masses repectively. Both antibodies, produced in rabbits, reacted with both vitellogenins in a competitive assay or by direct Vtg coating. For sexing of the fish, we finally chose the method of direct coating of serial plasma dilutions.