Protein ubiquitination is one of the most important post-translational modifications in eukaryotes, which controls many key fate-decision processes in cells from division to death. Ubiquitin dynamics and homeostasis are strictly regulated by E1-E2-E3 cascade enzymatic system which add ubiquitin to substrate proteins through activating, conjugating and ligating steps, and reversely regulated by deubiquitinases that release conjugated ubiquitin from substrate proteins. In human, there are approximately 100 deubiquitinating enzymes, 2 E1 enzymes, 40 E2 enzymes and more than 600 E3 ligases, which form a balanced but complex system remain to be unraveled. Chemical probes targeting ubiquitination systems and deubiquitinases have emerged as a powerful technique to profile these important proteins and especially extensively extend our knowledge about the molecular mechanism of ubiquitination process. This review aims to summarize basic design principles and synthetic methods for the chemical probes as well as their biological applications. In the first part, we define the composition of chemical probes including the reporter tag, recognition module and reactive group, which can be classified further based on the reaction principles of different reactive groups. Then we summarize previously synthetic and ligation strategies of recognition modules by total chemical synthesis and semi-synthesis, and detailed methods for the incorporation of reactive groups. In the second part, we highlight various biological applications for these probes. For profiling deubiquitinases, different scaffolds such as mono-ubiquitin and di-ubiquitin were designed based on their mode of action, with the hand of these probes, great progress has been made in the study of the multi-layer regulation of deubiquitinases controlling chain length, cleavage direction, linkage type, and substrate specificity. Furthermore, we discuss recent work that has brought a giant leap in the identification and/or mechanistic characterization of E3 ligases illuminated by activity-based chemical probes. Exquisite structural designs help to capture E3 ligases in different catalytic states and novel ubiquitin transfer mechanisms were visualized by structural biology. We anticipate great expansion of knowledge in catalytic mechanism of E3 ligases from these chemical probes, facilitating the theory-driven drug discovery and, in particular, paving the way for highly attractive technologies such as proteolysis-targeting chimeras (PROTACs).