The invention discloses a preparation method of plasmodium protein and application thereof in an anti-tumor aspect, and belongs to the technical field of biomedicine. The plasmodium protein contains an amino acid sequence shown as SEQ ID NO. 1. The protein has a certain killing effect on tumor cells, the inhibition rate on tumor cells HepG2 reaches 58.1%, which is close to the inhibition effect of25 mug/mL anti-tumor drug 5-Fu on tumor cells, and the protein has a certain clinic application prospect.