A method based on fluorescently labeled enzyme proteins was established to visualize the absorption properties of lipase at the oil-water interface, and it can be used for the effective observation of the distribution characteristics of lipase at the oil-water interface. The optimal conditions for observation include the following: oil content of 10-20% (wt%), concentration of fluorescently labelled enzyme protein of 0.25 mg/mL, reaction temperature of 25-30 °C, emulsion dispersion and stirring time of 10 min, and emulsion resting time of 30-120 s. Based on this method, a preliminary analysis of the effects of oil and lipase species on the distribution characteristics of lipase at the oil-water interface was performed. The results reveal that differences in the distributions of lipase at the oil-water interface of various fats and oils had a certain degree of correspondence with their specificity and that the distribution characteristics of the lipases on the surface of olive oil enabled effective catalytic hydrolysis to a certain extent. This method is a more objective guide for the development of lipase application technology in the fields of tanning, fur making, glue making, detergents, and sewage treatment and so on.