Alkaline protease was successfully immobilized onto amino-functionalized Fe3O4 nanoparticles. The enzyme loading was 388.2 mg of protein/g of support and the activity recovery was more than 54.2%. After immobilization, the affinity of alkaline protease toward substrate and its stability were significantly enhanced. The immobilized enzyme still retained 50.1% of its initial activity after 10 cycles of successive reuse, exhibiting excellent operational stability. The immobilized enzyme was capable of efficiently catalyzing hydrolysis of oat bran into oat polypeptides. Under the optimized conditions, the maximum DPPH radical scavenging rate (antioxidant activity) of oat polypeptides (8.4 mg/mL) was 82.3%, which was much higher than the reported result. Moreover, the prepared oat polypeptides by immobilized enzyme showed higher antioxidant activity than those prepared by free enzyme, owing to an increase of relatively hydrophobic components of oat polypeptides. Furthermore, the immobilized enzyme was demonstrated to be very promising for production of oat polypeptides on a preparative scale.