Objective  To optimize the chromatographic purification process of diphtheria toxoid (DT) by custom design. Methods  DT bulk purified by ammonium sulfate precipitation was concentrated, and then further purified by Sephacrayl S-300 (S-300) and Sepharose 4FF (4FF) gel filtration chromatography, respectively. Sampling volume, sample concentration, elution flow rate and chromatography medium were set as the experimental factors,while loading volume, purity and yield were set as the response values. A mathematical model was set up. The optimal parameters of gel filtration chromatography were confirmed, and the experimental results were validated. Results  The experimental results of the design showed that DT purity and yield obtained by S-300 gel filtration chromatography purification were higher than those obtained by 4FF. The optimal parameters of S-300 gel filtration chromatography were 2.5% for loading volume, 45 mg/ml for sample concentration, and 45 cm/h for elution flow rate. When S-300 gel filtration chromatography purification was performed with the optimal parameters, the verifying results of purification process met expectation. Conclusion  S-300 is more suitable as the gel chromatography medium of DT purification process than 4FF.