One of the tools for evaluating the reliability of diagnosis is external quality assessment scheme (EQAs), which is required the materials for expanding. Base on the results of survey the common samples and bacteria at the clinical microbiology laboratories [3J, the authors set up the process for manufacturing of EQAs samples (pus, sputum), which contain the malignant and contaminated bacteria, then the authors do the screening experiments, optimize them following the Plackett - Burman matrix, the steepest descent method, response surface methodology central composite design (RSM - CCD). Finally, the authors find out the condition for manufacturing and the ingredient with the best selecting factors for manufacturing EQAs samples containing the malignant and contaminated bacteria: pus (S. aureus, P. aeruginosa), sputum (Acinetobacter baumannii, P. aeruginosa, contaminated Streptococcus mitis).