The Vitellogenin (Vtg) gene sequence acts as an indicator to the fish reproduction, which can adapt to the environment factor or can influence the gonad development. The Vtg nucleotide sequence from Giant grouper was characterized using bioinformatics software. A homology search of the deduced amino acid sequence of the obtained Vtg DNAs (compare with 13 species) was carried out using the National Centre for Biotechnology Information website. Clustal W analysis was constructed a phylogenetic tree by using Molecular Evolutionary Genetic Analysis MEGA version 5.2.2. In order to verify the Vtg gene sequences obtaind and elucidate structure-function relationship in Vtg, by using DELTA BLAST of 3-D structure of Giant grouper with others fishes. Result of phylogenetic analysis using Maximum Likelihood (ML) and Neighbour Joining (NJ) showed tree analysis generated two separated tree topology. This similarity (0.015 distance matric viewer) was closely related in terms of their Vtg gene sequence although from different environmental and ecological conditions. In general, showed that Epinephelus lanceolatus Vtg is evolutionary more related to Poecilia latipinna. Epinephelus lanceolatus shows four main domains (Vitellogenin-N, DUF1943, DUF1944 and VMD), similarly found in Dicentrachus labrax but was different compare to Clarias macrochepalus, Catla catla, and Danio rerio. This indicates characteristic of Vtg domain for freshwater species is control by present of VMD in Vtg. The molecular approach can be done on Giant Grouper to understand the molecular respond towards fish growth and determine the individual of Giant grouper that has potential to increasing the Vtg production for increasing eggs quality.