The β-amino ketones produced through the Mannich reaction hold significant potential as candidates for various drugs. In this study, we optimized on-DNA Mannich reaction conditions and applied them to investigate the reactions of DNA-conjugated aldehydes with various amine and ketone building blocks. The developed on-DNA Mannich reaction preserved the DNA integrity and established viable routes for library production. These results underscore the potential of the Mannich reaction in DNA-encoded library (DEL) synthesis.