Vitellogenin, the egg yolk precursor protein, has studied as biomarker for sex identification of giant grouper (Epinephelus lanceolatus). Vitellogenin was synthesized successfully in liver of male giant grouper by implanted with 2 mg/kg of Estradiol-17-ß (E2). The levels of vitellogenin (vtg) in blood of E2-injected fish were comparable with those found in the blood of naturally male and female by observed with SDS-PAGE. With antigen from purified plasma E2-treated was produced polyclonal antibody of giant grouper after immunization process with rabbit for 72 days. Analyst by ELISA has demonstrated polyclonal antibody was developed within 2 weeks after primary immunization and greatest differences in absorbance at 1:8,000 dilutions. Validation by western blot, has verified vitellogenin as positive control (in female natural) and negative control (in male natural). It’s showed that the antigens vtg of giant grouper polyclonal antibody recognized the purified giant grouper vtg in natural female and treated estradiol male. The information on Vtg through this experiment could provide the basis of an immunological assay for sex identification and maturational status of the Giant grouper. Consequently, the aim of the present study was to establish a simple, efficient and non-lethal technique by using Vtg as biomarker indicator for rapid sex identification without killing or seriously injuring the sampled individuals.