In the present work, a procedure for leaching of lipoxygenase-II from soybean flour has been developed. The effect of operational parameters such as temperature, mixing speed, pH, sodium salts, leaching period, and reactor scale on enzyme extraction was studied. It has been found that analytical grade n-hexane is more advantageous for defatting of soybean flour than is industrial grade. Further, lipoxygenase-II was leached from defatted soybean flour by using sodium phosphate buffer of 0.05 M strength of pH 6.5. It has also been observed that by increasing the medium temperature from 4 to about 25 ± 1 °C, higher enzyme could be leached. The effect of a wide range of agitator speeds on the enzyme leached was investigated. It has been found that at 5 rps, a considerable amount of lipoxygenase-II could be leached. A vector diagram of the agitated tank and scale-up at 5 and 1.67 rps using CFD has been presented, respectively. Lsqnonlin Matlab version 6.5 was used to analyze the data statistically. A correlation has been developed.