CuAAC reaction is a well-known cycloaddition reaction between alkyne and azide. In this study, for monitoring intramolecular CuAAC reaction in aqueous buffered solution we synthesized a ratiometric fluorescent peptidyl probe which consisted of two pyrenes as a fluorophore attached to the peptides containing alkyne group and azide group. Since pyrene fluorophores are hydrophobic, for working of our probe in aqueous solutions, we introduced Arg, a hydrophilic amino acid which enhances the solubility of the probe. The mechanism involves reduction of Cu(II) to Cu(I) in presence of sodium ascorbate which catalyze cycloaddition reaction resulted in enhancement of monomer emission at 400nm and decrease of excimer emission at 480nm of peptidyl probe. Kinetic study of CuAAC reaction shows a first-order reaction which is totally different from previous reported results.  So, we investigated kinetic study of CuAAC under various conditions, such as different buffers, different metal ions, different pH, and different percentage of organic solvent and so on to determine whether the first-order reaction is due to the probe or due to the environmental factors or due to the change in molecular structure of the probe. Furthermore, experiments were carried out in RPMI media to make sure that it works well in media also. In media there were some delays in rate of reaction which was only in RPMI but not in aqueous buffer solution. To solve the problem, we carried out some reaction in presence of ligands which can enhance the rate of reaction.  Detailed study of this reaction could help us to compare the ability of various CuAAC ligands for improving reaction rate.