The use of Auxenochlorella pyrenoidosa (formerly Chlorella pyrenoidosa) and its intracellular substances (ISs) to promote biofloc development has been extensively studied. To identify the key components of the ISs of A. pyrenoidosa that drive biofloc formation, algal-extracted polysaccharides (AEPSs) and algal-extracted proteins (AEPTs) were isolated from the ISs. In this study, we established four groups: ISs, AEPSs, AEPTs, and tap water (TW, control), to investigate the effects of AEPSs and AEPTs on biofloc formation dynamics, water quality parameters, and microbial community composition. The results indicated no significant differences were observed between the ISs and AEPSs groups during the cultivation period. AEPSs significantly enhanced flocculation efficiency, achieving a final floc volume of 60 mL/L. This enhancement was attributed to the selective promotion of floc-forming microbial taxa, such as Comamonas, which can secrete procoagulants like EPS, and Pseudomonas and Enterobacter, which have denitrification capabilities. Water quality monitoring revealed that both AEPSs and AEPTs achieved nitrogen removal efficiencies exceeding 50% in the biofloc system, with AEPSs outperforming AEPTs. This is closely related to the fact that the microorganisms with increased flocculation contain numerous nitrifying and denitrifying bacteria. So, the intracellular polysaccharides were the key component of the ISs of A. pyrenoidosa that drive biofloc formation. These findings provide critical insights into the functional roles of algal-derived macromolecules in biofloc dynamics and their potential applications in wastewater treatment.