The biopolymer showed two protein-like fluorescence peaks (peaks A and B). Interactions of Pb(II) and biopolymer were quantified at various system pH values. System pH values significantly affect the quenching constant values for both peaks. Peak B plays a more important role in the interactions than peak A. Removal mechanism of metal ions by activated sludge system was further disclosed. The quantification and effects of system pH value on the interactions between Pb(II) and the biopolymer from activated sludge were investigated. The biopolymer had two protein-like fluorescence peaks (Ex/Em= 280 nm/326–338 nm for peak A; Ex/Em= 220–230 nm/324–338 nm for peak B). The fluorescence intensities of peak B were higher than those of peak A. The fluorophores of both peaks could be largely quenched by Pb(II), and the quencher dose for peak B was about half of that for peak A. The modified Stern-Volmer equation well depicted the fluorescence quenching titration. The quenching constant (Ka) values for both peaks decreased with rising system pH value, and then sharply decreased under alkaline conditions. It could be attributed to that the alkaline conditions caused the reduction of available Pb(II) due to the occurrence of Pb(OH)2 sediments. The Ka values of peak B were bigger than those for peak A at the same system pH values. Accordingly, the aromatic protein (peak B) plays the key role in the interactions between metal ions and the biopolymer.