Objective: In this study, the conditions of covalent immobilization of Pseudomonas fluorescens lipase onto an oxirane-activated support (Amberzyme) were optimized to obtain a high activity yield. Furthermore, the operational and storage stabilities of immobilized lipase were tested. Methods: Optimum conditions for immobilization were determined by changing individually the conditions (pH from 5 to 9; buffer concentration from 0.025 to 2.5 M; amount of Amberzyme from 100 to 500 mg and duration of immobilization from 24 to 120 h). Amounts of protein and the activity of enzyme were determined by UV/Vis (PYE UNICAM SP8-200 UV/Vis spectrophotometer). Results: Immobilization conditions (pH and molar concentration of immobilization buffer, enzyme/support ratio and immobilization duration) significantly affected the immobilization efficiency. 100% immobilization yield and 145% activity yield were achieved by optimizing the immobilization conditions. Operational and storage stabilities of immobilized lipase were determined as well. The immobilized enzymes retained its activity for 20 consecutive batch reactions. Furthermore, the immobilized lipase showed a high storage stability as no decrease in its activity was observed for 20 days. Conclusion: Our results obtained in the present study are the best in the covalent immobilization of Pseudomonas fluorescens lipase in the literature. Therefore our future studies will focus on using the immobilized Pseudomonas fluorescens lipase for the production of biodiesel, hydrolysis of oils and various important esterification reactions.