Histone ubiquitination plays critical roles in regulating the structure and dynamic of chromatin as well as DNA-driven processes. An understanding of how histone ubiquitin marks translate into diverse cellular events needs access to homogenously ubiquitinated histones. Here, chemical methods combining auxiliary-mediated and hydrazide-based native chemical ligation were developed to efficiently prepare several modified histones. Especially, we finish the first total chemical synthesis of di-ubiquitin modified histone. The synthetic histones were successfully applied for reconstitution of nucleosomes and further biochemical or structural analysis.