The disclosure relates to a preparation method of eel polypeptide flavor extract, the eel polypeptide flavor extract, and the eel sauce. The preparation method of the eel polypeptide flavor extract includes the following steps: obtained by using eel processing by-products, eel cutting off the main meat tissues, successively performing microbial fermentation, Maillard reaction and extraction and separation processes. The eel polypeptide extract obtained above is used to making a condiment eel sauce. The eel sauce is nutritious and convenient, it can be used directly for table eating or served as cooking condiments to enhance the aroma and taste of dishes and increase the attractiveness of dishes. The disclosure realizes a new technological research on the preparation of polypeptide and flavor products, opens up a scientific utilization and processing method of seafood processing by-products, and provides a new way for making full use of such resources.