Abstract: Neutrase AS1.398 could be used to separate soybean protein and oil from soybean powder in reverse micelles.Revealing the enzyme catalysis mechanism could help to optimize the extraction technology.The reaction mechanism of the enzyme in AOT/iso-octane reverse micelles was investigated with soybean protein isolated as the substrate.Since the dispersion of substrate molecule showed Poisson distribution in the reverse micelles, then through calculating the ratio of the number of substrate molecule to that of the enzyme, it was deduced that the probability of single substrate molecular reaction was predominant.Single substrate molecular enzyme catalysis reaction considering molecular exchange effects in reverse micelles was proposed with aqueous system reaction as reference, the Michaelis-Menten constant in aqueous system was about ten times greater than that in the reverse micelles from the model.The experimental values of the constants were 3.2×10^-3g•ml^-1 and 4.2×10^-4g•ml^-1,respectively.The experimental results were in good agreement with those calculated by the model.It showed that the model can express the enzyme catalysis reaction in the reverse micelles.