This work proposes a simple, cost-effective, and efficient preparation of a composite made from magnetic diatomaceous earth (mDE) coated with polyaniline (mDE@PANI). The material was used as a matrix for immobilizing industrial enzymes: invertase, β-galactosidase, and trypsin. The mDE@PANI was characterized by several methods, and the results suggested that the nanoparticles were approximately 12 nm in size, exhibited superparamagnetic behavior, and displayed a good magnetic response and that magnetite comprised the main iron oxide phase. Moreover, several studies were conducted for all immobilized derivatives, including determination of optimal pH and temperature; kinetic parameters; thermal stability and reusability. The obtainment of three novel magnetic biocatalysts with superior performance (in terms of activity and stability) compared to their free counterparts demonstrated the efficacy of the mDE@PANI nanoparticles. In addition, the enzymatic derivatives can be easily recovered from the reactor by using an external magnetic field. Finally, the present methodology allowed the achievement of good mDE@PANI matrix together with three promising magnetic biocatalysts with several potential biotechnological applications.