Lysine fermentation broth was desalinated by electrodialysis to reduce hardness concentrations as a pretreatment procedure for the purification and recovery of amino acids. Electrodialysis performance was investigated in terms of the rate of reduction in conductivity in dilute solutions and electrodialysis cell resistance for different ion exchange membranes at a constant current density. Among the membranes investigated in this study, membranes with high water content showed the better performance for hardness removal. Fouling experiments revealed that organics gave rise to fouling effects on the anion exchange membrane during demineralization of the lysine fermentation broth. The pulsed electric field with the half-wave power enhanced the electrodialysis performances by mitigating membrane fouling in desalination of the lysine fermentation broth. This study successfully demonstrated the potential use of pulse power as an effective cleaning-in-place (CIP) method during electrodialysis of fermentation broth.