Objective In order to prepare ubiquitin samples with different chain types, different chain lengths and phosphorylation modifications.Methods This study mainly uses the biological enzymatic method to describe the preparation routes of the above samples. There are two main methods, the first method is to add ubiquitin monomer one by one and then extend the ubiquitin chain; the second is to prepare mixed polyubiquitin chains through an enzymatic reaction, and then purify and separate these mixed chains.Results The experimental results show that both methods can achieve the purpose of preparing polyubiquitin chains. Further, phosphorylated ubiquitin samples were prepared by phosphorylation of ubiquitin. K11/K48 branched chain ubiquitin was prepared by K11 and K48 ubiquitinase.Conclusion In short, based on the above preparation route of the ubiquitin chain, it is possible to further perform post-translational modifications such as phosphorylation modification on different subunits of different link forms. The site-specific attachment of a prosthetic probe and isotope labeling on different subunits allowed us to perform NMR and FRET measurements. In summary, these methods presented here can provide reference and help for scientists involved in Ub research.