Laboratory-scale sequencing batch reactors (SBRs) were operated on activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Polyphosphate-accumulating microorganisms (PAOs) play an important role during the enhanced biological phosphorus removal (EBPR) process. Fluorescence in situ hybridization (FISH) was applied to assess the proportions of microorganisms in the sludge. The aim of this study was to optimize hybridization of PAOMIX and RHC439 probes by orthogonal design. Orthogonal optimization test of the four factors were conducted under the individual three levels. The optimal hybridizition conditions were as follow: hybridization temperature 46°C, hybridization time 2.5h, washing time 15min, formamide concentration 35%(PAOMIX probe); hybridization temperature 50°C, hybridization time 2.5h, washing time 20min, formamide concentration 20% (RHC439 probe).