Sludge dewatering has proven to be an effective method to reduce the volume of sludge. In this study, a novel stratification approach aimed at better understanding the factors influencing the sludge dewaterability (as determined by capillary suction time, CST) was developed. The sludge flocs from 14 different full-scale wastewater treatment plants (WWTPs), including sewage, leachate, industry, and special-source sludge, were stratified through centrifugation and ultrasound into five layers: (1) supernatant, (2) slime, (3) loosely bound extracellular polymeric substances (LB-EPS), (4) tightly bound EPS (TB-EPS), and (5) pellet. The results showed that the distribution pattern of proteins (PN) in the sludge flocs differed from that of polysaccharides (PS). The normalized CST correlated with PN (R2 > 0.72, p < 0.01) and PN/PS (R2 > 0.51, p < 0.01) in the supernatant, slime, and LB-EPS, but not with PN and PN/ PS in the pellet and the sludge flocs as a whole or with PS in any of the fractions and or the sludge flocs as a whole. The results suggest that PN and PN/PS in the supernatant and slime layers, which are usually decanted due to their assumed lower content of organic matter, markedly impact sludge dewaterability.