The invention discloses an alfalfa-trypsin inhibitor MT-mth2-89i19 as well as an encoding gene and application thereof. The alfalfa-trypsin inhibitor MT-mth2-89i19 provided by the invention is as thefollowing protein of a), b), c) and d): a) a protein with an amino acid sequence shown as a sequence 2; b) a fusion protein obtained by connecting an N terminal and/or C terminal of the protein shownas the sequence 2 with a tag; c) protein with the same function, obtained by substitution, deletion and/or addition of one or more amino acid residues of the amino acid sequence shown as the sequence2; and d) a protein with the homology being 75 percent or above with the amino acid sequence shown as the sequence 2 and with the same function. Shown through experiments, the protein has very good inhibition effect to therioaphis trifolii, has the inhibition effect to both of the survival rate and the reproductive rate of aphids and can be used for preventing and controlling the aphids.