Objective Amino acids, vitamins and inorganic salts were used to replace beef extract and peptone to develop a selective medium with controllable quality for Salmonella. Methods Effects of 20 kinds of amino acids, 11 kinds of vitamins or 4 kinds of inorganic salts on the growth characteristics of Escherichia coli ATCC 25922 were studied, and the optimal concentration was determined. The beef extract and peptone in bismuth sulfite (BS) agar or Hektoen Enteric (HE) agar were replaced, and the chemically defined BS agar or HE agar was developed. Results Adding asparagine, aspartic acid, valine, thiamine hydrochloride or riboflavin to the basic medium was the most favorable for the growth of Escherichia coli ATCC 25922, and the OD₅₉₅ were all higher than 0.1. However, adding serine, lysine hydrochloride, isoleucine, nicotinic acid, biotin or disodium hydrogen phosphate to the basic medium did not promote the proliferation of Escherichia coli, and the OD₅₉₅ was almost 0. The chemically defined BS agar or HE agar was prepared by replacing beef extract, peptone with 35 kinds of nutrients according to the optimal concentration. After verification of quality control strains, chemically defined BS agar or HE agar was similar to commercial medium in cell growth index, colony morphology and special color reaction. Conclusion Chemically defined BS agar or HE agar meet the quality requirements of national standard.