Purified nattokinase has the potential to be developed as functional food additives and drugs to prevent or treat several diseases. Arginine modified magnetic nanoparticles to isolate nattokinase from Natto broth were developed in this study. Functional nanoparticles were prepared by covalently conjugating cationic ligands onto the surface via a carbonyldiimidazole coupling method. The arginine-modified magnetic nanoparticles demonstrated the 2.4-fold adsorption of nattokinase compared with the lysine modified nanoparticles. The behavior of the adsorption and desorption for nattokinase onto surface-modified magnetic nanoadsorbents from the natto solution was systematically investigated. The results of transmission electron microscopy indicated that surface modification on nanoparticles did not affect size distribution of magnetic nanoadsorbents. The adsorption kinetics was fitted by the pseudo-first-order and pseudo-second-order equations. It was found that the pseudo-first-order rate equation was the best one for characterizing the adsorption kinetics. In this study the purification factor, the maximum adsorption capacity, and recovery yield were 8.0, 3.79U/mg nanoparticles, and 15%, respectively. Overall, our results demonstrated that the arginine-coated magnetic nanoadsorbents could efficiently adsorb nattokinase from the Natto broth.