Enhanced biological phosphorus removal (EBPR) is the main phosphorus removal technique for wastewater treatment. During the anaerobic-aerobic alternative process, the activated sludge experienced the anaerobic storage of polyhydroxy-β-alkonates (PHA) and aerobic degradation, corresponding the infrared peak intensity of sludge at 1 740 cm-1 increased in the aerobic phase and declined in the anaerobic phase. Compared with PHA standard, this peak was indentified to attribute the carbonyl of PHA. The overlapping peaks of PHA, protein Ⅰ and Ⅱ bands were separated using Gaussian peak fitting method. The infrared peak area ratios of PHA versus protein I had a good relationship with the PHA contents measured by gas chromatography, and the correlation coefficient was 0.873. Thus, the ratio of the peak area of PHA versus protein Ⅰ can be considered as the indicator of the PHA content in the sludge. The infrared spectra of 1 480~1 780 cm-1 was selected, normalized and transferred to the absorption data. Combined with the chromatography analysis of PHA content in the sludge sample, a model between the Fourier-transform infrared spectroscopy (FTIR) spectra of the sludge and PHA content was established, which could be used for the prediction of the PHA content in the unknown sample. The PHA content in the sludge sample could be acquired by the infrared spectra of the sludge sample and the established model, and the values fitted well with the results obtained from chromatograph. The results would provide a novel analysis method for the rapid characterization and quantitative determination of the intracellular PHA content in the activated sludge.