Abstract: This study aimed to explore the importance of dissolved organic nitrogen (DON) in the aquatic ecosystem and its ecological effects, and to find a scientific method to evaluate the bioavailability of DON and to provide a basis for further revealing the mechanism of lake eutrophication. Three-dimensional fluorescence excitation-emission matrix (3DEEM-PARAFAC) was used to estimate sediment DON bioavailability. Various components were identified by PARAFAC model according to the fluorescence excitation emission spectra. Surface sediment samples from different regions of Erhai Lake were selected to study the difference of DON content variation by comparing the cultures inoculated with bacteria and algae under room conditions. The variation characteristics for DON structures during incubation were characterized by UV-visible absorption and 3DEEM spectra. The results showed that:(1) Bioavailability of sediment DON incubated by algae (8.49%-42.5%) was slightly lower than that by bacteria (10.5%-45.3%). (2) Under algae incubation, the DON bioavailability had significant positive correlation with the growth of algae cell density, indicating algae growth could reflect sediment DON bioavailability. (3) The correlation between DON content and its spectroscopy characteristic parameters of 3DEEM, SUVA₂₅₄ and S_R during the incubation by bacteria was better than that by algae. SUVA₂₅₄ had different declining degree, and S_R showed different increasing degree, indicating DON with high molecular weight and aromaticity could be degraded by microorganisms. Compared with terrestrial PARAFAC components, autochthonous components showed higher bioavailability. (4) Compared with bacteria incubation, algae incubation method could reflect the influence of sediment DON on algae growth more directly. It could be used as a method to measure the DON bioavailability of lake sediments to some extent. In conclusion, it is more suitable to evaluate the Erhai sediment DON bioavailability by algae incubation.