In this study, a thermo-tolerant and alkaline lipase enzyme was purified from Lactobacillus brevisand immobilized onto modified ?-Fe3O4florisil nanoparticles (?-Fe3O4MF NFs) and the usability of free lipase (FL) and immobilized lipases (IML) as detergent additives was investigated. Lipase enzyme was purified by fractional precipitation using 20% ammonium sulfate, DEAE-Sephadex ion-exchange chromatographic column, and Sephacryl S200 gel filtration chromatographic techniques. Then, the enzyme was purified, which resulted in 135.2-fold purification. Its molecular mass was determined to be 57 kDa by SDS-PAGE. The covalent immobilization of purified lipase was done using ?-Fe3O4MF NPs. ?-Fe3O4MF NPs and IML were characterized by using SEM, TEM, FT-IR, and XRD. IML showed a good thermo-stability and its activities were calculated as 80% at 60°C. The free and IML enzymes were most stable at alkaline pHs in the range of 7.0–10.0. Also, IML is more stable towards metal ions compared to free lipase enzyme. Washing performances of some detergent formulations were investigated in the presence and absence of Lipase. Olive oil was removed by the detergent alone and by the detergent and IML at ratios of 45% and 72%, respectively. The study on removal of oil stain from cotton cloths indicated that the removal of oil was superior in the presence of IML and IML with detergent, when compared to the detergent alone.