Polypeptides are fascinating materials with unique properties for various biological materials. We highlight here recent advances in amino acid N-carboxyanhydrides (NCAs) and synthetic polypeptides from the aspects of chemistry, self-assembly and biological applications. New synthetic methodologies, mechanistic studies and optimization of polymerization conditions for the preparation of well-defined novel polypeptides are comprehensively reviewed and evaluated. Functional polypeptides, mostly prepared from novel NCA monomers, with ultra-stable helical conformation, stimuli-sensitive properties, or glycoprotein mimetics are summarized. We also highlight a number of interesting self-assembled structures of polypeptides in solid state and solution, with particular emphasis on those structures other than amphiphilic self-assembly. The biological applications of polypeptides in drug and gene delivery are also reviewed. Future directions and perspectives are discussed in the conclusion.