The microbial flocculant (MFX) extracted from Klebsiella pneumoniae J1 was used to remove carbamazepine in prepared wastewater and domestic sewage. The influence factors and flocculation mechanism were studied. The optimal carbamazepine removal conditions for MFX were pH of 7-8, 7 mL of flocculant, 0.1 mL of coagulant, and 35°C, and the removal rate reached 81.75%. MFX was efficient in the removal of carbamazepine in both domestic sewage (75.03%) and secondary sedimentation tank effluent (69.76%). The pseudo-first-order kinetic equation fitted the adsorption process better than the pseudo-second-order kinetic equation, which suggested that the adsorption was not pure chemical adsorption. The analysis of floc size suggested that the repulsive force between carbamazepine and MFX was weakened under alkalescent conditions, which can help the growth and coherence of flocs and increase the carbamazepine removal efficiency. Enough dosage of MFX can generate larger flocs, but excessive dosage of MFX will decrease the carbamazepine removal rate because of increase in electrostatic repulsion. The analysis of 3D-EEM and FTIR suggested that hydroxyl, amino, and carboxyl in MFX played an important role in the removal of carbamazepine. As an eco-friendly and highly efficient microbial flocculant, MFX has potential for practical applications in carbamazepine removal.