The invention discloses oyster pallium hexapeptide with an effect of promoting osteogenesis as well as a preparation method and application of the oyster pallium hexapeptide. The amino acid sequence of the oyster pallium hexapeptide is Phe-Phe-Glu-Pro-Lys-Phe. Fresh oyster mantle is used as a raw material, pepsin and trypsin are used for hydrolysis and centrifugation to obtain supernate, then ultrafiltration is carried out to obtain filtrate with the molecular weight being 1000 Da or below, finally freeze drying is carried out, the sequence of oyster hydrolytic peptide is obtained through mass spectrum identification, and by means of a molecular docking technology and a database prediction simulation method, the oyster hydrolytic peptide is obtained. Oysterpeptide with high osteogenesis promoting capacity is screened from a plurality of peptide sequences. The oyster hexapeptide obtained by the invention has the effects of improving osteoblast proliferation and differentiation capacity and improving dexamethasone-induced zebra fish bone loss, and can be used as a beneficial dietary supplement in related products for delaying bone loss.