This study aim was to optimize the manufacturing conditions polyethylenimine-coated polysulfone/bacterial biomass composite fiber (PEI-PSBF) to remove anionic pollutants from aqueous solution. The contents of biomass, PEI, and glutaraldehyde (GA) were selected as independent variables, and the response was defined as Reactive Yellow 2 (RY2) uptake. The manufacturing conditions were optimized by response surface methodology (RSM) with the full factorial central composite design (CCD). The determined coefficient of determination (R2) value of the reduced quadratic model was 0.9551, and the optimal manufacturing conditions were predicted as 4.145 g of biomass, 1.104 g of PEI and 3.9 μL of GA, at where the predicted RY2 uptake was 543.78mg/g. For validating the RSM-predicted results, the RY2 sorption capacity of the optimized PEI-PSBF was evaluated through isotherm experiments. The experimentally confirmed maximal uptake was comparable to predicted one. From these studies, the manufacturing conditions for PEI-PSBF were well optimized and its sorption capacity was 3.83 times higher thanthat of the PSBF.