A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 degrees C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 degrees C, did not lose any activity over 48 h at 25 degrees C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.