Despite great advancement in genetic typing, phenotyping is still an indispensable tool for categorization of bacteria. Certain amino acids may be essential for bacterial survival, growth, pathogenicity or toxin production, which prompts the idea that the intrinsic ability to utilize single amino acid under live-or-die situation could be a basis for differentiation of bacteria species. In this study, we determined the single amino acid consumption profiles of 7 bacterial species, and demonstrated that most bacteria have species-specific pattern of amino acid consumption. We also discovered that bacterial strains from different hosts, toxigenicity, and antibiotic-resistance presented distinct preference for certain amino acids. Taken altogether, the amino acid consumption profiles showed potential to be a novel tool complementary to study not only bacterial categorization but also biochemical characteristics of the bacteria such that its phenotyping can be used to uncover strategies for nutritional, pharmaceutical, taxonomic, and evolutionary aspects of bacterial researches.