The biological treatment of wastewater generates a substantial amount of waste sludge that requires dewatering before final disposal. Efficient sludge dewatering is essential to minimize storage and transportation costs. In this study, the sludge conditioners polydimethyldiallylammonium chloride (PDMDAAC) and ferric chloride (FeCl3) were sequentially dosed, and the pH was adjusted to 3. As a result, the sludge moisture content (MC) was reduced to 59.4%, achieving deep dewatering. After conditioning, the tightly bound extracellular polymeric substances (TB-EPS) were reduced from 34.5 to 10.2 mg g-1 VSS, with the majority of the reduced fractions being composed of protein (PN). In contrast, soluble EPS increased more than 8 times. Subsequent studies revealed that the decrease in PN from TB-EPS primarily involved tryptophan and tyrosine proteins, accompanied by a significant reduction in the N-H and C[double bond, length as m-dash]C absorption peaks. These results highlight the critical role of TB-EPS dissolution in achieving deep dehydration, with the N-H in PN was identified as the key group influencing sludge dewatering. Combined with the changes in sludge particle size and morphology, the dewatering mechanism can be summarized as follows: PDMDAAC dissolves TB-EPS, simultaneously disrupting the floc structure and refining the sludge. Subsequently, FeCl3 reconstructs these elements, forming larger particle sizes. Finally, hydrochloric acid reduces TB-EPS once again, releasing bound water. This study offers alternative methods and new insights for achieving deep dewatering of waste sludge.