The invention provides tuna red meat ACE inhibitory peptide and a preparation method thereof, and relates to the field of preparation of ACE inhibitory peptide. The peptide fragment amino acid sequences of the tuna red meat ACE inhibitory peptide are Met-Trp-Asn, Met-Glu-Lys-Ser, Met-Lys-Lys-Ser or Leu-Pro-Arg-Ser. The preparation method comprises the following steps of (1) unfreezing the tuna redmeat, performing drying and performing degreasing for standby application; (2) performing enzymolysis on red meat powder after pretreatment to obtain enzymatic hydrolysate; (3) performing enzyme deactivation treatment on the enzymatic hydrolysate to obtain enzyme deactivation enzymatic hydrolysate, performing freeze drying to obtain polypeptide powder, and testing ACE inhibitory activity; and (4)subsequently, performing ultrafiltration, performing column chromatography, performing high efficiency liquid phase chromatographic purification, and performing amino acid sequencing to prepare the tuna red meat ACE inhibitory peptide. The prepared tuna red meat ACE inhibitory peptide is good in inhibitory activity, low in preparation cost, high in efficiency and suitable for industrial production.