Inspired by diversity-oriented synthesis, we have developed a series of DNA-compatible transformations utilizing on-DNA vinyl azide as a synthon to forge divergent N-heterocyclic scaffolds. Polysubstituted imidazoles and isoquinolines were efficiently obtained with moderate-to-excellent conversions. Besides, the “one-pot” strategy to prepare in-house on-DNA vinyl azides afforded synthons readily. Results from substrate scope exploration and enzymatic ligation further demonstrate the feasibility of these N-heterocycle syntheses in DNA-encoded chemical library construction.