Fresh neem leaves (Azadirachta indica A. Juss) consist of quercetin (flavonoid) compound, responsible for yellow colour as a natural dye. The study investigated the variable optimization in fresh neem leaf extraction process and its application. The optimization was conducted using response surface methodology (RSM) and ANOVA test. The coefficients of determination (R2) were generalized at 96.46 and 96.39 % for optimizing extraction process and application process parameter of natural dyes from neem leaves, respectively. The optimum variables for the extraction process were obtained at 50 % w/v feed-solvent ratio, the temperature of 80 ºC and extraction duration of 80 min with the response concentration of 46.19 g/L. Furthermore, the optimum variable on the application of natural dyes from neem leaves was recorded when alum was applied as fixator as much as 14.09 g/L and Turkish red oil (TRO) as mordant at 11.16 g/L resulted the response of 0 % on the degree of faded.