The invention specifically relates to a hard clam polypeptide, and a preparation method and application thereof, belonging to the field of marine biotechnology. The preparation method comprises the following steps: subjecting the soft body of a hard clam to homogenization at first and then to centrifugation, adding ethanol into the obtained supernatant until ethanol content is 60 to 80%, carrying out standing overnight, then carrying out centrifugation and taking a supernatant. Experimental results show that the hard clam polypeptide has good anti-oxidation and anti-ageing effect. The preparation method provided by the invention can make full use of the abundant resources of hard clams to prepare the hard clam polypeptide with strong anti-oxidation and anti-ageing effect. The hard clam polypeptide provided by the invention can be used for preparation of drugs with anti-oxidation and anti-ageing effect or health products with anti-oxidation and anti-ageing effect, and has a wide application scope and good economical and social effect.