Riboflavin Binding Protein (RBP) is isolated and purified for the first time from a single egg of Ostrich (Struthio camelus). The 238 amino acid sequence of this protein is determined using 2DE and Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Peptide Mass Mapping (PMM). The amino acid sequence of the riboflavin binding protein showed 95.8% sequence homology with the RBP isolated from the egg of Emu (Dromaius novaehollandiae), belonging to the same Ratite family. Ostrich egg RBP protein sequence showed homology to a lesser extent with the other avian species. Further, the protein modification sites such as glycosylation, N-myrystoylation sites were identified using Scan Prosite tool.