Proteins require many additional chemistries beyond the natural 20 amino acids that have limited number of functional groups to perform their natural functions. Genetically encode adding an amino might enable the evolution of proteins and entire organisms with new or enhanced properties. In fact, we can make an unnatural amino acid with the steric and electronic characteristics and it is possible to introduce a probe function, PTM, metal chelator, Photoaffinity label or chemical moieties to the desired position in the proteins. This technology allows the production of a modifying or labeling protein and, probing the protein to study protein structure and function, and modulating the activity of the protein and was able to identifying of proteins. Single-molecule FRET can provide an ideal analytical method for understanding the structure and dynamics of molecules and interaction of biomolecules. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. To solve this problem, we developed a general and facile method to the labeling position as specific proteins using incorporation of the two different amino acid (AlK.AcF) Therefore, using a newly evolved using Alkyl lysine-specific aminoacyl-tRNA synthetase / tRNA CA and p-acetylphenylalanyl-tRNA synthetase / tRNACUA, We were able to incorporation of AcF, AlK to 34 113 position of calmodulin, and then through the hydrazone formation and click reaction using the click , we could labeling the dye with a high efficiency. The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics. In addition, we implemented bio-orthogonal reaction in mammalian cells through the incorporation of unnatural amino acids. Technique for labeling a protein in mammalian cells can be observed for localization of the protein, or the protein monitoring. So it will be able to provide an important clue to find the function of intracellular protein are not known in the existing and to establish an interaction between the proteins. strain-promoted alkyne-azide cycloaddition reaction is suitable for mammalian cell experiment. The reason is that there is no cell toxicity, have fast reaction rate and takes place in a mild condition. Using this reaction, we were successfully biosynthetic the tubulin protein as the site-specific labeling. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling or GFP labeling. Therefore, it is expected that there may be more applicable to study protein which is involved in most biological processes, an important function.