This invention belongs to the field of biomedical technology, specifically relating to a method for preparing bioactive polypeptides and their applications. This invention prepares bioactive polypeptides from donkey-hide gelatinprotein through an enzymatic hydrolysis reaction. The bioactive polypeptides possess iron-chelating ability, enhancing Hb and RBC production in a mouse anemia model. Therefore, the polypeptides of this invention have blood-nourishing and blood-tonifying functions, are non-toxic, have no side effects, and also have advantages such as simple preparation, easy absorption, and ease of widespread application.