Ubiquitination is an important protein post-translational modification process in eukaryotic cells, participating in a variety of cellular functions such as protein degradation and signaling pathways. The polyubiquitination modification of the substrate proteins is a continuous process, which not only involves the complex participation of enzymes related to ubiquitin system, but also refers to more complex structural interactions and ubiquitin chain assembly mechanisms. Ubiquitin chain modifications determine the fate of the downstream substrate proteins. The crucial role of ubiquitin-conjugating enzyme E2 in the formation of ubiquitin chains has attracted more and more attentions. Understanding of the mechanisms of ubiquitin chain formation is conducive to the discovery of disease targets and treatment related to the ubiquitin system. In this review, we summarize the research progress of the mechanisms of ubiquitin chain formation, involving how E2 reacting with different types of E3 determine the type and the assembly process of ubiquitin chain formed on the substrates, with complex structural information provided. Two different mechanisms on how ubiquitin chain assembly, including the sequential addition mechanism and the en bloc transfer mechanism, are also discussed.