Adsorption has demonstrated great promise for removing dissolved organic nitrogen (DON), but effective solutions to alleviate the competitive adsorption of different types of DON are lacking. Herein, a novel sequential adsorption technology is designed for DON fractional removal from actual biotreated pharmaceutical wastewater. Three carbon xerogels (CXs) are customized via precisely controlling the gel process and constructing the macropore (CX5.4, 163−647 Å)−mesopore (CX6.2, 80−212 Å)−micropore (CX6.8, 17−136 Å) sequential adsorption column. The sequential adsorption column exhibits superior performance compared with that of single CX columns, which can effectively remove DON (82.3%), reduce acute toxicity (87.4%), and have suitable regeneration ability (14.7% decreased after three cycles). Analysis of DON removal characteristics [molecular weight (MW) and molecular composition] reveals that CX5.4 preferentially removes the high-MW DON via hydrogen bonding or electrostatic interactions, alleviating irreversible regeneration. CX6.8 enhances the removal of the low-MW and high-unsaturated DON molecules via pore filling and π−π interactions, further decreasing toxicity. Due to the coexistence of the adsorption mechanism described above, CX6.2 is the main DON adsorption unit. Moreover, the CX sequential adsorption column is more cost-effective than activated carbon. Overall, this study provides an effective and sustainable technology for DON adsorption treatment in actual wastewater.