Genetic code expansion (GCE) technology has enabled more than 200 chemically and structurally diverse non-canonical amino acids (ncAAs) to be site-specifically introduced to proteins with high specificity and fidelity. This technology has allowed us to study biological processes including structure, catalysis, transport, and defense at a new level of molecular precision. Despite the massive success of GCE technology, there are still deficiencies that restrict its utility. First, high level of ncAA supplement is required for stop codon suppression, which is ineffective and eco-unfriendly. Second, there are only limited chemical functionalities that could be incorporated into proteins by GCE. Third, lots of reported ncAA only showed the genetic incorporation without demonstrating applications in solving actual biological questions. In this dissertation, we describe the above limitations of GCE technology and provide new insights and progressions. First, multiple “completely autonomous” species that possess the biosynthetic and translational machinery for making proteins that contain the 21st amino acid: O-methyltyrosine have been generated. We demonstrated that the endogenous biosynthesis of ncAA reaches higher intracellular ncAA concentration than attained through exogenous feeding, leading to greater genetic incorporation efficiency. Importantly, we showed that the limited bioavailability of exogenously fed ncAAs in multicellular systems can also be overcome by endogenous biosynthesis. The advantages of the autonomous system not only allow for enhanced efficiency of ncAA incorporation but also open new opportunities in multicellular systems for encoding ncAAs with poor bioavailability. Second, to expand the chemical toolbox of GCE technology, we designed and synthesized an isocyanide-containing ncAA: ε-N-2-isocyanoisobutyryl-lysine (NCibK). A mutant pyrrolysine tRNA synthetase HibK-1RS/tRNAPyl CUA pair that specifically recognize NCibK was identified. Several bioconjugation reactions at both small molecule and protein level have been demonstrated, showing the robustness of the methodology. The ease of synthesis, versatility of reactivity, and good compatibility of isocyanides make NCibK an attractive ncAA for future biological applications. Third, we demonstrate new applications for the reported ncAA 4-fluorophenyl lysine (FPheK) to resolve current limitations in cancer therapeutics and its production. A methodology to synthesize a bispecific small molecule - antibody conjugate from a commercially available antibody without protein engineering was developed. This methodology provides a powerful platform to generate bispecific agents by conjugating small-molecule-like ligands targeting different membrane markers to human antibodies, or to generate various kinds of antibody drug conjugates (ADC). In summary, the work in this dissertation has shown advances in every aspect of current obstacles in non-canonical amino acid technology.