Histone ubiquitination affects the structure and function of nucleosomes. Here, we reported a one-pot synthesis of ubiquitinated histone analogues using 1,3-dibromoacetone (DBA) as the cross-linking reagent. The key step is that under the acidic borate buffer, the DBA linker can be efficiently installed to the Cys of the recombinant Ub mutant, followed by the coupling between the Ub-DBA with histones at physiological pH. The process requires a single HPLC step or orthogonal affinity tag purification to obtain ubiquitinated histone at about 24-38 mg/L expression.