Nitrogen fixation is one of the most important external sources of new nutrients in the oligotrophic oceans, which supports up to 50% of new production in the North Pacific. However, the cycling of the fixed N among particulate and dissolved phases in the euphotic zone and its regulation mechanisms remain poorly understood. In this study, we aimed to investigate the partition of diazotroph-derived nitrogen in the marine dissolved organic nitrogen (DON) revealed by the isotopic analysis and molecular characterization. Onboard nitrogen fixation incubations were conducted with spiked ¹⁵N₂gas at the North Pacific Subtropical Gyre stations. The δ¹⁵N ratios and high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in the solid phase extracted DON (SPE-DON) during the experiments were analyzed simultaneously. The δ¹⁵N ratios of the SPE-DON showed a positive correlation with the nitrogen fixation rates (NFR), which indicated the partition of fixed nitrogen in the dissolved phase. The FT-ICR-MS data were analyzed and screened for the nitrogen-containing compounds that were involved in the nitrogen fixation process. A formula assignment working flow was developed in the analyzed samples with different NFR and spiked ¹⁵N ratios. This work provides further evidence and insights into the fate of diazotroph-derived nitrogen at molecular levels.