The formation of recalcitrant Dissolved Organic Matter (DOM) pools in the ocean has been a longstanding challenge due to the chemical complexity of DOM. Linking nitrogen sequestration and microbial reworking via the production of recalcitrant Dissolved Organic Nitrogen (DON) molecules remains elusive. Here, we characterized intricate molecular composition of DOM using FT-ICR-MS, with a particular emphasis on DON, across three representative regions in the tropical eastern Indian Ocean. Our findings demonstrated the microbial origin of DON in epipelagic waters, with ammonia-oxidizing archaea exerting important control on the enrichment of peptide-like compounds. Microbial respiration was identified as a key driver of DOM transformation throughout the water column. This process enhanced the recalcitrance of DOM and DON by generating molecules with high levels of unsaturation and oxidation, characterized by low bioavailability. These effects were most pronounced in the equatorial region, which demonstrated an exceptional capacity to accumulate nitrogen-rich compounds through microbial processing, thereby facilitating to long-term nitrogen sequestration. Furthermore, we provided a valuable dataset representing microbially derived recalcitrant DON. Our study highlights that a small fraction of DOM with comparatively higher bioavailability is selectively preserved, though the majority of DON persists in the deep ocean due to its recalcitrant nature. This work provides novel molecular-level insights into microbially derived recalcitrant DON molecules, and holds significant implications for the detailed interpretation of global nitrogen sequestration.