The algal uptake of dissolved organic nitrogen (DON) in the anaerobic–anoxic–oxic (A2O) process was investigated in this study. Anaerobic, aerobic and effluent DON samples from two wastewater treatment plants (WWTPs) were separated into hydrophilic and hydrophobic fractions using a DAX-8 resin coupled with an anion exchange resin and a nanofiltration (NF) pretreatment. Hydrophilic DON accounted for 66.66%–88.74% of the entire DON for the two plants evaluated. After a 15-day incubation, 16.95%–91.75% DON was bioavailable for algal growth, and untreated samples exhibited higher DON bioavailability, with 52.83% DON average uptake rates, compared with the hydrophilic and hydrophobic fractions (45.53% and 44.40%, respectively) because the pretreatment caused the inorganic salt to be resistant to algae. Anaerobic untreated samples, hydrophilic fractions and hydrophobic fractions showed higher DON reduction rates and higher biomass accumulation compared with the other DON fractions due to the decomposition of resistant organics by anaerobic and anoxic bacteria. DON in aerobic and effluent samples of plant A was more bioavailable than that of plant B with usages of 27.49%–55.26% DON. DON bioavailability in the anaerobic–anoxic–oxic process decreased in the following order: anaerobic>effluent>aerobic. The DON contents were reduced after anaerobic treatment in the two plants. The EEM-PARAFAC model identified three DON components, including two humic acid-like substances and one protein-like substance in plant A and two protein-like substances and one humic acid-like substance in plant B.