This paper aimed to examine how the amphoteric conditioner poly (acrylamide‐′acryloyloxyethyl trimethyl ammonium chloride −2‐acrylamido‐2‐methyl‐propane sulfonate) (PADA) with different anionic degrees (AD) affected the properties of sludge flocs in the conditioning. The floc properties were characterized by morphological parameters (floc size distribution, fractal dimension, specific surface area, and pore volume), physical properties (floc strength and surface charge density), and chemical constituents (Fe3+, Al3+ and extracellular polymeric substances (EPS), including the polymeric proteins and carbohydrates). The results of this investigation revealed that (1) morphological properties of flocs were associated with anionic degree, particularly in the range of 0–4%, where the anionic degree led to a shift of the particle size toward groupings of larger diameter, meanwhile better regularity and increased compactness of floc structure formed. (2) The introduction of the anionic groups indeed had bad effects on flocs in terms of its stability and charge neutralization, but the downtrend could be inhibited by adjusting the anionic degree of polymer to a reasonable level. (3) The dissolved EPS for sludge followed a role of decreasing firstly then increasing with increased anionic degree, but the content of bound EPS kept nearly constant. (4) The polymer with anionic groups had apparent effects on enrichment of metal ions. POLYM. ENG. SCI., 57:197–205, 2017. © 2016 Society of Plastics Engineers