Ubiquitylation, also termed ubiquitination, is one of the most important post-translational modifications in eukaryotic cells. It is a process by which a small signaling protein, called ubiquitin composed of 76 amino acids, is conjugated to protein substrates via an E1-E2-E3 enzymatic cascade. Ubiquitin can be attached to lysine, serine, threonine and cysteine residues of its substrates. Ubiquitin itself contains seven lysine residues, therefore, ubiquitylation can form various polyubiquitin chains to produce complex ubiquitin codes. Ubiquitylation can alter the fates of ubiquitylated proteins including kinase activation, alteration of protein localization and proteolysis via the 26S proteasome and is involved in nearly every aspect of biological activities in eukaryotic cells. Recent studies indicated that more complicated post-translational modifications can also be found on ubiquitin including sumoylation, ubiquitylation, phosphorylation and acetylation. These modifications largely increase the complexity of ubiquitin signals. Ser65 of ubiquitin is the first characterized phosphorylation site whose biological functions have been extensively studied in human cells. It has been shown that Ser65 phosphorylation by PINK1 kinase is critical for the activation of Parkin ubiquitin ligase during mitophagy induction. The researches on Ser65 phosphorylation of ubiquitin boosted the studies on biological significance of the rest phosphorylation sites of ubiquitin. Now it is clear that phosphorylation of Ser57 residue is involved in endocytosis and stress responses, including oxidative stress in yeast, whereas phosphorylation of Thr12 and Thr66 residues plays important roles in DNA damage response. In the case of Ser57 residue, members of the AMPK-related kinases phosphorylate it, however, mechanisms by which Ser57 phosphorylation regulates endocytosis or oxidative stress response are still unclear. Also, no experimental evidences are available in mammalian system yet. One interesting fact is that many AMPK-related kinases contain a ubiquitin-associated domain (UBA), although some studies suggested these UBA domains do not possess any binding capabilities to polyubiquitin chains. However, some of these kinases could phosphorylate the Ser57 residues on the M1 polyubiquitin chain, implying these UBA domains do recognize certain polyubiquitin chains. In this review article, we summarized the post-translational modification sites of ubiquitin, especially phosphorylation sites and highlighted the biological functions of Ser65, Ser57, Thr12, Thr66 phospho-ubiquitin proteins. We also discussed alternations of biophysical properties brought by the phosphorylation of ubiquitin. Finally, we proposed a few future research directions related to the phosphorylated ubiquitin.