A series of biodegradable amphoteric chitosan-based flocculants (3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CTA) modified carboxymethyl chitosan, denoted as CMC-CTA) with different substitution degrees of CTA were prepared successfully. The content of carboxymethyl groups in each CMC-CTA sample was kept almost constant. The solubility of the various flocculants showed that, higher cationic content of flocculants caused a better solubility. The flocculation experiments using kaolin suspension as synthetic water at the laboratory scale indicated that the substitution degree of CTA was one of the key factors for the flocculation properties. With the increase of cationic content, the flocculants were demonstrated better flocculation performance and lower dosage requirement. Flocculation kinetics model of particles collisions combining zeta potential and turbidity measurements was employed to investigate the effects of the cationic content of the flocculants on the flocculation properties from the viewpoint of flocculation mechanism in detail. Furthermore, flocculation performance using raw water from Zhenjiang part of Yangtze River at the pilot scale showed the similar effects to those at the laboratory scale.