The E2-Ub-nucleosome conjugation chemistry has emerged as a practical tool for investigating the mechanisms of E3-catalyzed ubiquitination of nucleosomal histones H2A and H2B, but its application to histones H3 and H4 remains to be explored. Here, we describe the development of the first synthetic H3-based E2-Ub-nucleosome conjugate to trap the intermediate during E3 PHF7-catalyzed H3K14 ubiquitination. Through chemical protein synthesis, we generated H3K14-Ubch5c-Ub and assembled the H3K14-Ubch5c-Ub-nucleosome conjugate. Electrophoretic mobility shift assays (EMSA) revealed that the H3K14-Ubch5c-Ub-nucleosome forms a stable complex with PHF7, resulting in an 8-fold increase in binding affinity compared with that of an unmodified nucleosome. Chemical crosslinking mass spectrometry (CX-MS) analysis of the PHF7-Ubch5c-Ub-nucleosome complex revealed a working model in which the RING domain of PHF7 interacts with the nucleosomal acidic patch, thereby anchoring the E2 catalytic center to the H3K14 modification site. Overall, this work broadens the scope of the E2-Ub-nucleosome strategy and establishes a methodological foundation for the mechanistic study of E3-mediated H3 ubiquitination.