•A highly selective and sensitive IC method was developed and validated.•Only a single valve and cation-trapping column were used for condition optimization.•20 amino acids and 7 sugars were separated simultaneously without co-elution.•The method was applied to the medium of Clostridium thermocellum successfully.•The work built a new analysis platform for water-soluble metabolites.An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5–4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.