The emergence of non-canonical amino acids (ncAAs) has enriched the functional pool inherent in canonical amino acids (cAAs). ncAAs are important building blocks in the production of various pharmaceuticals. The biosynthesis of ncAAs has emerged as an alternative to traditional chemical synthesis because of its environmental friendliness and high efficiency. The breakthrough genetic code expansion (GCE) technique developed in recent years has allowed for the incorporation of ncAAs into target proteins, giving them special functions and biological activities. Biosynthesis of ncAAs and their incorporation into target proteins within a single microbe has become an enticing application of such molecules. Based on that, in this study we first review the biosynthesis methods for ncAAs and analyze the difficulties in biosynthesis. We then summarize the GCE methods and analyze their advantages and disadvantages. Further, we review the application progress of ncAAs and anticipate the challenges and future development directions of ncAAs.