The invention relates to a penaeus japonicus anti-oxidative peptide. The amino acid sequence of the penaeus japonicus anti-oxidative peptide is shown as Gly-Asn-Gly-Leu-Pro. The invention further relates to a preparation method of the penaeus japonicus anti-oxidative peptide. The method comprises the following steps: raw material pretreatment; ultrasonic-assisted enzymolysis; ultrafiltration treatment; gel column treatment; reversed-phase high-performance liquid chromatography treatment. Compared with the prior art, the penaeus japonicus anti-oxidative peptide has higher antioxidant activity; the head of penaeus japonicus is taken as a raw material, so that the raw material source is wide, the cost is low, and the problem of production of leftovers in the processing process of the penaeus japonicus is solved; the penaeus japonicus anti-oxidative peptide has theoretical significance to further increase in the economic value of the penaeus japonicus; moreover, ultrasonic treatment and protease hydrolysis are combined, so that the hydrolysis rate is increased, effective components in the raw material are extracted fully, and the extraction rate is increased; moreover, the extraction manner is mild, and the effective components to be extracted are prevented from being damaged; meanwhile, the penaeus japonicus anti-oxidative peptide with a reliable structure can be obtained through multiple separation and purification, and gradual sieving.