Four different configurations of capacitive deionization (CDI) were assembled to comparatively investigate the contribution of different ion exchange membranes (IEMs), including anion exchange membrane (AEM) and cation exchange membrane (CEM), to the enhancement of desalination performance. The results showed that the low charge efficiency of membrane-free biochar-based CDI system was mainly attributed to co-ion repulsion rather than Faraday reactions, and a single IEM was not effective in suppressing the co-ion repulsion effect thereby exhibiting a similar low charge efficiency. Compared with AEM, CEM was more beneficial for high coulombic efficiency by restraining the oxygen reduction reactions. In addition, the electrosorption and desorption behaviors of the biochar-based membrane capacitive deionization (MCDI) under different operating conditions were explored. The results showed that zero voltage desorption (ZVD) was unfavorable to the desorption capacity of the electrode, especially in high concentration solutions, the reverse voltage desorption (RVD) led to higher desorption efficiency but required higher energy consumption. Therefore, energy consumption and regeneration efficiency need to be a trade-off in practical applications to realize energy saving and efficient desalination.