•An improved method to eliminate the persulfate effect for carbohydrate analysis.•Sodium sulfite is the best pretreatment chemical to eliminate the persulfate effect.•The improved method is accurate and reliable based on the results of actual samples. Advanced oxidation processes (AOPs) based on activated persulfate have received extensive attention for sludge pretreatment. Persulfate (PS) is a persistent oxidant and the excess persulfate in the sludge affects the carbohydrate analysis using the traditional phenol-sulfuric acid method. In this study, an improved phenol-sulfuric acid method was developed for the determination of carbohydrates in the presence of persulfate, and a critical procedure was introduced before the conventional determination with the assistance of the chemical pretreatments. The best reagent was screened from four reductants (i.e. Na2S, KI, Na2SO3 and Na2S2O3) and two free-radical scavengers (i.e. phenol and benzoic), and the optimal conditions were determined based on the monosaccharides (i.e. glucose, galactose and mannose) and the polysaccharides in different EPS fractions from two activated sludges. The results showed that Na2SO3 was the best reagent for chemical pretreatment due to redox reaction, high selectivity and colorless oxidation product, and the others were not suitable as pretreatment reagents mainly since the reaction products affect the final spectral analysis. Besides, the optimal Na2SO3 concentration for eliminating the impact of PS was 150 mmol/L, and the relative deviation of measurement is less than 5% if the PS concentration was on more than 100 mmol/L, regardless of the kinds of carbohydrates. Compared with the anthrone-sulfuric acid method and the sulfuric method, the improved phenol-sulfuric acid method has a great advantage for determining the carbohydrate concentration in the presence of PS.