The phosphorylation of ubiquitin significantly enhances the complexity of the ubiquitin code. However, the molecular consequences of ubiquitin phosphorylation at threonine residues remain largely uncharacterized. In this study, we present an effective method for the total chemical synthesis of threonine-phosphorylated ubiquitin, producing tens of milligrams of all six in vivo-identified threoninephosphorylated ubiquitin analogues: pUbT7, pUbT12, pUbT14, pUbT22, pUbT55, and pUbT66. The biochemical activities of phosphorylated ubiquitin analogues were examined in vitro. Our results show that threonine phosphorylation has a differential impact on E2 charging, with phosphorylation at residue Thr7 exhibiting significant inhibition. In addition, threonine phosphorylation significantly affects the E1-E2-E3-mediated assembly and deubiquitinase-mediated disassembly of polyubiquitin chains in a site-specific manner. Collectively, this work provides new insights into the effect of phosphorylation on the ubiquitin code.