For development of an on-line monitoring unit of fermentation process, an auto dilution unit based on traditional chemical and biological analytical method was developed and the performance was evaluated. The dilution unit was constructed with two syringe pumps and flow direction change valves and fully automated. Total delivery volume of two pumps using distilled water was measured to confirm the operating stability And diluted concentrations of three substrate solutions (glucose, lactic acid, ethanol) were compared with a standard method with a high performance liquid chromatograph (glucose, lactic acid) and gas chromatograph (ethanol). Relative error values of total delivery volume of the pumps were below 3% and standard deviation values were 0.003 (n=5). Relative error values of diluted concentration of the dilution unit measurements were below 2% with 1/10 of dilution ratio and 70, $80{mu}{ell}$ of sample volume conditions for glucose and lactic acid, 1/30 of dilution ratio and $70{mu}{ell}$ of sample volume conditions for ethanol, respectively. In case of the ethanol, cause of the evaporative characteristics, the relative error values showed over 5% whole experimental conditions.