The invention discloses a preparation method of immobilized recombinant homosapiensarginase. The preparation method comprises the following steps that S1, a first covalent carrier and a second ion carrier are pretreated; S2, the recombinant homosapiensarginase is treated; S3, the pretreated first covalent carrier and the recombinant homosapiensarginase are sufficiently mixed, and an immobilizationreaction is carried out on the recombinant homosapiensarginase; S4, the pretreated second ion carrier is added into a reaction liquid, and the immobilization reaction is carried out; S5, the obtainedimmobilized recombinant homosapiensarginase is drained for liquid replacement or freeze-dried for preservation. The preparation method has the advantages that the influence of an existing immobilization method on the enzymatic activity is changed, the loss of the enzymatic activity is reduced, and the problem that an immobilized enzyme is easy to contaminate is solved; the invention aims at providing industrial application of the immobilized recombinant homosapiensarginase. The immobilized recombinant homosapiensarginase is utilized to catalytically produce L-ornithine, and the industrial application prospects are good.