A new method was described for simultaneous determination of Z. mobilis metabolites by reversed phase-high performance liquid chromatography (RP-HPLC). The samples were analyzed on a C18 column (Waters SunFireTM C18, Φ 416mm×150mm, 5μm), and the mobile phase was 10mmol/L pH2.5 KH2PO4 solution with a flow rate of 0.8mL/min. Under this optimized condition gluconate, pyruvate, lactic acid and acetic acid were completely separated and determined with a photodiode array detector (PAD). The IC-PakTM on-Exclusion column(50A ,7μm,7.8mm×300mm) were used and the mobile phase was 10mmol/L sulfuric acid solution with flow rate of 0.6mL/min. Glucose, sorbitol, succinic acid, acetic acid and ethanol were simultaneously determined by refractive index detector(RID). The standard deviation values and the average recovery rates were in the range of 0.005%-0.018%, 96.86%-101.99%, respectively.