Abstract: Proteins in the human body are usually made of 20 natural amino acids.Through different amino acid combinations and isomerization, proteins of diverse functions are built.An emerging genetic code expansion technology can introduce unnatural amino acids into specific sites of target protein, endowing the protein with new biological characteristics including covalently binding with proximal proteins, carrying fluorescence, and mimicking specific protein post-translational modifications.In this paper, based on the structure and function of unnatural amino acids, the applications of different types of unnatural amino acids in regulating protein''s stability, studying protein''s conformation, expression level, and localization, and uncovering heretofore unknown protein-protein interactions were reviewed.Besides, genetic code expansion of unnatural amino acids is anticipated to find broad utilities in biomedicine by bringing new ideas and methods to the design and optimization of biologics.