Ubiquitination and SUMOylation interactions between proteins play a critical role in various biological processes and the progression of diseases. To investigate the biological functions of the direct crosstalk between protein ubiquitination and SUMOylation biochemically, we have developed a method for synthesizing Ub-tagged SUMO2 dimers using an expanding genetic code approach. In this description, we outline the procedures for creating Ub-SUMO heterodimers through the incorporation of defined noncanonical amino acids (ncAAs) and biorthogonal functional group-guided conjugation techniques. These homogeneous Ub-SUMO heterodimers will serve as valuable tools for studying the mechanisms of Ub-SUMO chain elongation, degradation, topology, and other related protein–protein interactions.