Abstract: With the antarctic krill by-product as raw material,the optimal hydrolysis process was determined by single factor and orthogonal experiments,and free amino acids were detected in antarctic krill peptone,the optimum concentration of inoculated microorganisms was got and their culture effect was compared. The results showed that the optimal hydrolysis conditions for the preparation of Antarctic krill peptone were as follows:temperature 60 ℃,pH5.0,after adding alkaline protease for 1.5 h,then adding flavour protease to continue hydrolysis for 3.5 h,adding 1% of total enzyme,compound enzyme ratio was 1:1.The hydrolysis rate under this condition was 28.38%. Antarctic krill peptone was rich in 17 free amino acids with a total free amino acid content of(2968.83±35.1) mg/100 g. The optimum concentrations of E.coli,Staphylococcus aureus,Bacillus subtilis,Bacillus cereus and cultured in antarctic krill peptone solution were 3.5,5.0,4.0,4.5 g/L. The OD₆₀₀ of the four microorganisms in the beef cream peptone was only about 8% larger than that in the antarctic krill peptone. It was indicated that antarctic krill peptone can replace beef cream peptone as a component of microbial culture medium.