Investigation of the optimum conditions for the combined application of Saccharomyces sp. (yeast) and Leuconostoc sp. (lactic acid bacteria, LAB) isolated in a previous study to the development of novel sourdough was carried out by response surface analysis. First, the cell growth conditions were analyzed. LAB showed good proliferation under conditions of 30-$35^{circ}C$, low pH, and high acidity, whereas the growth of yeast was inhibited. The growth of yeast was optimum at $25^{circ}C$ for 24 h. Based on these results, analysis of sourdough was carried out by varying the LAB population, temperature, and time after fixing the number of yeast. It was determined by response surface analysis that the optimal conditions for fermentation are LAB population of $10^5$ CFU/mL, temperature of $25^{circ}C$, and reaction time of 24 h. From these results, the growth of LAB should be constantly maintained, and an appropriate pH that does not inhibit the growth of yeast due to the presence of generated organic acid is required to allow for the unique properties of sourdough. This study could give useful information for the development of novel sourdough.