A promising whey protein separation technique, Reverse micelles (RM) extraction could be an important tool for obtaining the valuable food ingredients. RM techniques have the potential to continuously extract specific proteins from an aqueous mixture, achieving simultaneous concentration and purification of specific proteins in an efficient manner. The extraction of lactoferrin (LF) protein, using nano-sized reverse micelles of nonionic surfactant polyoxyethylene p-t-octylphenol (triton-X-100) is attempted here. Suitability of reverse micelles of cationic surfactant cetyl trimethylammonium bromide (CTAB) and triton-X-100/CTAB mixture in organic solvent for LF extraction is also investigated. The raw milk treated with 1M HCl at pH 6.5 and centrifuged to get whey sample. The whey sample was concentrated with 0.1µm membrane enrichment and diluted five times to its original volume with 50 mM phosphate buffer at pH 6.35 containing 100mM sodium chloride. The aqueous solution was then mixed with an equal volume of organic solvents containing the surfactant. The aqueous phase and reverse micelles phase has been separated by centrifugation and the protein is accumulated in the aqueous phase. The extract was fractionated using ammonium sulphate and purity improvements are done by HIC (Hi Trap Phenyl) column and followed by IEX(DEAE cellulose) column chromatography which was previously equilibrated with phosphate buffer with the pH maintained at 7.8 and eluted by increasing ionic strength with sodium chloride. The results show that the LF evolves more in CTAB than in CTAB+ tritonX-100 mode which may be due to reduced hydrophobic interaction.