An improved, simple, effective and superior protocol has been developed to immobilize amano lipase from Pseudomonas fluorescens on woolen cloth using polyethyleneimine (PEI) with glutaraldehyde (GA) cross-linking. The success of immobilization was confirmed by FTIR and confocal laser scanning microscope (CLSM), the latter proving that enzyme is well distributed across the wool fiber surfaces throughout the cloth. Woolen cloth therefore provides a large outer and inner fiber surface area for immobilization with minimal mass transfer resistances during immobilization. The optimal protocol (GA at 0.5% and pH 6, lipase solution pH 6) gave an enzyme load of 46.6mgg dry cloth with expressed activity of 178.3U, 46.8% immobilization yield and 30.2% retained activity. Zeta potential measurements showed that PEI significantly enhanced the positive charge on woolen cloth and shifted the isoelectric point to approximately 7. Therefore at a lipase solution pH of around 6, the wool-PEI and lipase are oppositely charged, leading to a maximal adsorption of lipase to the wool surface. The immobilized lipase also had a good stability and 81% of its original activity was maintained after 10 runs in tributyrin emulsion hydrolysis. This protocol provides a significant improvement in terms of retained activity and lipase stability compared to previous immobilizations on wool and opens up the possibility of using wool as a cheap and effective lipase support material for continuous lipase reactions/reactors and possibly enzyme enhanced woolen fabrics.