Dissolved organic nitrogen (DON) is actively involved in N cycling and transformation processes in the ocean, yet how this large N pool is formed remains elusive. In this work, we incubated ¹⁵N-labeled individual amino acids (Ala, Val, and Phe) and short peptide (Ala-Val-Phe-Ala, AVFA) in seawater and monitored their fates over 21 – 60 d. Ca. 25 – 45% of Phe-N and Val-N were transformed to “uncharacterizable DON”, as compared to only about 6% for Ala-N, indicating that the percentage/amount of DON formed depends on specific amino acids. Through a stable isotope probing approach, 5 Phe-derived DON molecules from the incubations were found to be present in natural waters, and their possible structures were proposed. These results shed new lights on the formation mechanisms of refractory DON, including the roles of specific amino acids and particular chemical structures that may resist decomposition in millennial time scales.