Abstract: This study aimed to establish a new method for simultaneous determination of sucrase and inulinase activity using ion chromatography. Sucrose was used as the substrate and enzymatic hydrolysised by sucrase. Sodium borohydride solution was applied for converting glucose and fructose to mannitol and sorbitol. Afterwards, sucrase was inactivated at high temperature, and inulinase was added to get the mixed solution containing mannitol, sorbitol, glucose and fructose. To determine the concentration of four compounds in the mixed solution, chromatographic separation was carried out using CarboPaC^TM PA1 250 mm×2 mm column with water, sodium hydroxide and sodium acetate as gradient eluents (the flow rate of 1.0 mL/min and the column temperature of 30 ℃). Finally, sucrase activity was calculated based on the total amount of mannitol and sorbitol, and inulinase activity was calculated according to the total amount of glucose and fructose. The results showed that mannitol, sorbitol, glucose and fructose could be determined within 20 min. All the linear correlation coefficients were all above 0.9995, and resolutions were above 1.0. Thus, sucrose could be used as substrate for the determination of inulinase activity. Glucose and fructose could be completely converted into mannitol and sorbitol after adding 1.2 mL of 10 mg/mL sodium borohydride solution in a water bath at 60 °C for 30 min. The activities of sucrase and inulinase determined by this method did not show significantly differences compared to 3,5-dinitrosalicylic acid colorimetry determination method (DNS method) (P>0.05). The relative standard deviation of sucrase activity and inulinase activity determined were 2.0% and 1.7% respectively. The method did not involve toxic and harmful reagents used in traditional methods. Therefore, this method could be used to determine the activities of sucrase and inulinase simultaneously, and would provide a new idea for the determination of other enzyme activities.