Two series of binary graft cationic starch-based flocculants (CS-DMCs and CS-DMLs) with different hydrophilicity and charge density (CD) were prepared by graft copolymerization of acrylamide with 2-(Methacryloyloxy)-N,N,N-trimethylethanaminium chloride and methacrylic acid 2-(benzyldimethylaminio) ethyl chloride, respectively, on the starch (St) backbone. The sludge dewatering performance of CS-DMCs and CS-DMLs were evaluated and compared based on the changes in filter cake moisture content (FCMC), specific resistance of filtration (SRF), fractions and components of extracellular polymeric substances, and various physiochemical characteristics of sludge flocs and cakes. Increase in CD of the St-based flocculants caused improved sludge dewaterability. Under the similar CD, CS-DML with relatively high hydrophobicity exhibited lower FCMC and SRF, larger and denser sludge flocs, and better permeability of sludge cakes than CS-DMCs due to the synergistic effects of charge neutralization, bridging flocculation and hydrophobic association. Furthermore, a second-order polynomial model on the basis of phenomenological theory was successfully applied to quantitatively evaluate the influences of the two important structural factors of these St-based flocculants, i.e., hydrophobicity and CD, on the sludge dewaterability. The structure–activity relationship of the St-based flocculants in sludge dewatering was obtained according to the theoretic simulation. The dewatering mechanisms was discussed in depth on the basis of the experimental and simulated results; besides, the FCMC and optimal dose can be predicted by the established structure–activity relationship. This current work offered a novel and valuable way to exploit and design of low-cost and high-performance graft natural polymeric flocculants applied in efficient conditioning of sludge.