Abstract The kinetics for the tributyrin hydrolysis using lipase ( Pseudomonas fluorscenes CCRC-17015) were investigated in the liquid–liquid and liquid–solid–liquid reaction systems in a batch reactor. The lipase was covalently immobilized onto the surface of porous polymethylacrylamide (PMAA) crosslinking with N, N-methylene biacrylamide with a spacer of ethylenediamine actived by glutaraldehyde. The conditions such as tributyrin concentration, temperature, agitation, and pH value, were evaluated to achieve the optimum reaction conditions for both free lipase and immobilized lipase. The kinetic parameters in the reaction system were also obtained for two reaction systems. The turnover numbers calculated for free lipase and immobilized lipase were 29 and 5.7 s −1, respectively. The parameters of k and k m obtained using Lineweaver-Burk plot method were 26.2 mol/(mg min) and 1.35 mol/dm 3 for free lipase, 5.2 mol/(mg min) and 0.2 mol/dm 3 for immobilized lipase, respectively. The experimental results revealed good thermal stability, with greater stability at higher pH value for immobilized lipase in the liquid–solid–liquid reaction.