A simple method was developed to recover high P content fraction in enhanced biological phosphate removal activated sludge. The method was based on the concept of centrifugal separation in sucrose solution with different density. The quinone profiles of the fractionated samples were analyzed to determine quinone biomarkers for phosphate-accumulating organisms. Addition of 4% paraformaldehyde and lowered temperature around 4°C protected the activated sludge from lysis due to the osmotic pressure of the solution. Homogenization for 15 min was appropriate for the dispersion of the flocs by a homogenizer (φ2cm, 3500rpm). Using 50.0%, 51.2% and 52.4% sucrose solutions, the activated sludge was fractionated appropriately. In the case of 51.2% sucrose solution, relatively high P content activated sludge and low P content one were fractionated. Comparing their quinone profiles, the high P content one had higher fractions of Q-8 and MK-7.