We report on the development of a functionalized membrane-based technology for selective enrichment of milk fat globules from raw bovine milk. Functionalization was conducted by in situ polymerization of acrylic acid within a polyvinylidene fluoride membrane, followed by the electrostatic attachment of a cationic polymer to impart a net positive charge. The functionalized membrane-based technology enabled a one-step method of selective separation of globules directly from milk-based on size and charge. The presence of globules in the eluate was confirmed by fluorescence microscopy. Quantification of the extracted phospholipids from globules in the eluant revealed a significantly higher amount of polar lipids than the permeate. Our study describes a comprehensive analysis of selective enrichment of fat globules using a functionalized membrane and demonstrates the beneficial effect of extracted phospholipids from enriched fat globules.