Replication-coupled DNA methylation maintenance by DNMT1 is mediated with dual monoubiquitinated PCNA-associated factor 15 at K15 and K24 (PAF15Ub2), but the molecular mechanism of DNMT1 activation by PAF15Ub2 remains unclear. Here, taking advantage of chemically synthesized PAF15Ub2, we determine the active conformation of DNMT1 by Cryo-EM. With an optimized convergent synthetic route, the 262 amino acid protein PAF15Ub2 was obtained in high purity by total chemical synthesis. Six peptide segments were efficiently assembled and converted to the native sequence of PAF15Ub2 by employing one-pot ligation techniques and selective deselenization chemistry. Since the synthetic PAF15Ub2 possesses native isopeptide bonds, cryo-EM analysis was conducted under a reducing condition that prevents undesired aggregation of DNMT1 and is not compatible with previously developed dislufide-linked PAF15Ub2 mimic. Furthermore, methyltransferase activity of DNMT1 was stimulated by the synthetic PAF15Ub2, suggesting the successful reconstitution of DNMT1 activity in vitro.