The invention discloses a preparation method of a peptide-calcium chelate and application thereof. The raw materials are subjected to low-temperature ultrafine-smashing treatment, the reaction solid-liquid ratio and raw material utilization ratio can be improved, the utilization ratio of proteins and dissolution of peptides in the raw materials are improved by using a two-stage enzymolysis technology, the product stability and flavor are improved by using a deodorization, decoloration and impurity removal technology, calcium is self-transformed by using a fishbone hydrolysis and chelation technology to improve the bioavailability of the calcium, and the product storage and transportation cost is effectively reduced through a concentration and spray-drying technology. The preparation methodof the peptide-calcium chelate and the application thereof have the advantages that the high-valued development of by-products of fish processing can be achieved, the nutritive value of products is increased, and the application field is expanded.