Abstract A flow-injection system utilizing the Trinder reaction for the determination of glucose is designed and optimized. Glucose is converted to gluconic acid by passing it through a single-bead-string reactor (SBSR) onto which glucose oxidase enzyme has been immobilized. As it flows through a second SBSR, the gluconic acid reacts with a reagent stream of horseradish peroxidase, 4-aminoantipyrine and 3,5-dichloro-2-hydroxyphenyl sulfonate. The absorbance of the quinoneimine dye produced is then monitored at 510 nm in a flow-through cell. Optimum operating conditions were sought by using both univariate and Composite Modified Simplex procedures. Seven variables were considered. The performance of the system was improved by a factor of 22.5 relative to the starting conditions. A calibration curve obtainedat the optimum conditions was linear for 0–3.3 mM glucose and usable for 0–5.5 mM glucose. The optimization procedures revealed some interesting aspects of the Trinder reaction. scatter diagrams generated from the simplex data showed definite trends for each of the seven variables. These are discussed.