Milk microfiltration has gained attention in the dairy industry due to its ability to selectively separate and concentrate bioactive components from milk and its further application in food fortification. One such compound is milk fat globules, a bioactive component with potential health benefits, which undergoes structural and functional damage, membrane phospholipids loss, experience longer processing time, and higher recovery cost due to its current multi-step separation process. We developed a single-step approach based on size for the separation of smaller-size milk fat globules from cow and buffalo milk using a porous polysulfone membrane through a cross-flow microfiltration system. An asymmetric polysulfone membrane with an average pore size of 0.8 ± 0.03 µm was synthesized via phase inversion and subsequently characterized for surface morphology, hydrophilicity, porosity, thermo-mechanical properties, and water contact angle. The membrane demonstrated good performance, with initial milk permeate flux ranging from 296 to 613 L/m2h at different transmembrane pressures (e.g., 0.2, 0.4, 0.6 bar) for both animal groups, with a gradual decline due to concentration polarization, and eventually reaching a steady state. Notably, at lower transmembrane pressure, the membrane exhibited excellent antifouling properties (flux recovery of 81.56–83 %) and reusability over three cycles. Microscopic examination of milk fat globules confirmed the successful size-based separation with intact globule membrane, high phospholipid yield, and mass balance. The results, to the best of our knowledge, demonstrate for the first time an in-depth analysis of membrane fouling and reusability for selective isolation of milk fat globules.