A stable insoluble P450 BM3 system that can be easily separated from the reaction medium for recycling, while using less-expensive cofactors (NADH and BNAH) than costly NADPH, can represent a promising biocatalyst in industrial applications. In this context, the present work investigates the immobilization of double mutant cytochrome P450 BM3 (R966D/W1046S) by adsorption and cross-linking-adsorption on Ni2+-functionalized magnetic nanoparticles (MNPs). By oxidizing NADH or BNAH, the immobilized BM3 succeeded in hydroxylating the substrates (10-pNCA and myristic acid) to a similar degree as the free enzyme. The adsorbed enzyme showed 88% hydroxylation residual activity after five reaction cycles (five continuous days), which was increased to 100% by cross-linking the adsorbed enzyme. In addition, the cross linked adsorbed enzyme kept 41% of its initial activity toward NADH after one month of storage at 4 degrees C, while the free enzyme showed only 31% residual activity after 1 week and was inactive afterward. The results of this work highlighted that the appropriate choice of the enzyme-support-cofactor system can result in an active,stable, and recyclable biocatalyst, which could attract growing industry interest.