To address the problem of dewatering treatment of river and lake sediment, biological enzymes such as common lysozyme, α-amylase and neutral protease were used for conditioning. Correspondingly, the influence of biological enzyme content, mud concentration, and conditioning time on the dewatering performance of sediment were investigated. Furthermore, the moisture content of the mud cake and the specific resistance of the sludge after biological enzyme treatment were measured to investigate the dewatering effects of river and lake sediment. The results showed that the three selected enzymes could effectively improve the dewatering performance of river and lake sediment, and their conditioning effects were in the order of α-amylase> neutral protease>lysozyme. Notably, when the α-amylase content was 2 mg/g, the mud concentration was 11% and the conditioning time was 0.5 h, the moisture content of the sediment was reduced by 31.72%, and the corresponding specific resistance was reduced by 167×109 cm/g. Scanning electron microscopy (SEM) results showed that after the treatment the particle size of the sediment became smaller and the intergranular pores increased by size and number, which in turn significantly increased the water passages in the mud, thereby improving the dewatering performance of the sediment.