Amino acids and small peptides are important active constituents and metabolites of microorganism. With the progression in modern analytical techniques, the qualitative and quantitative analysis methods of amino acids and small peptides from microorganism also keep improving. In this paper, the pretreatment methods were discussed from the quenching, extracting, derivatization and concentration steps. Furthermore, the properties and practical application of the commonly used separation and detection methods were discussed. Meanwhile, the prospects of the analysis methods for amino acids and small peptides from microorganism were also highlighted.