Cytochromes P450 are a vast and important class of heme monooxygenases with a broad range of substrates and corresponding functions. Decades of studies attest that enzymatic processes with many members of P450s, mostly in drug metabolism, display complex kinetic behaviors depending on substrate, cofactor, enzyme mutation, ionic strength of the reaction medium, and so forth. However, kinetics of enzymatic processes with P450s remain to be elucidated from different aspects, particularly in the case of immobilized enzyme systems. A notable member of P450s is the soluble and self-sufficient P450 BM3 from Bacillus megaterium which hydroxylates saturated/unsaturated carbon chains at room temperature and acts as an ideal model to study atypical behavior of mammalian P450s. In this context, this study investigates the kinetic behavior of a P450 BM3 in its free and immobilized form [the enzyme was immobilized on surface-modified magnetic nanoparticles (MNPs) by three different methods: adsorption, cross-linking-adsorption, and covalent bonding] within the 10-(4-nitrophenoxy)-decanoic acid (10-pNCA) hydroxylation process [cofactors: nicotinamide adenine dinucleotide (NADH) or N-benzyl-1,4-dihydronicotinamide (BNAH)]. The enzymatic hydroxylation showed sigmoidal behavior independent of the enzyme concentration or cofactor, and consequently a three-parameter non-Michaelis-Menten kinetic model with cooperative binding of substrate to the active site (Hill coefficient of 2) was used to correlate the experimental data. Noticeably, comparable hydroxylation efficiency was observed among the adsorbed, cross-linked-adsorbed, and free BM3, suggesting that the kinetic model developed for the free enzyme can be appropriate for the mentioned immobilized BM3 systems.