Interfacial tension is an important physical property affecting the droplet formation process in microfluidic devices. This work presents the variation of dynamic interfacial tension caused by slow adsorption of surfactant, as well as its influence on the liquid/liquid microdispersion process in a T-shaped microchannel. Using hexane/water-Tween 20 as the working system, it was observed that the droplet size changed with the variation of surfactant concentration when the concentration of Tween 20 was lower than 10 mmol/L, but hardly changed at higher concentrations, which was caused by the unsaturated adsorption and saturated adsorption of surfactant, respectively. The saturated interfacial tension was measured with an interfacial tension meter, and the relationship between the interfacial tension and the droplet diameter was established. Accordingly, the dynamic interfacial tension with unsaturated adsorption of surfactant was determined. The main factors affecting the dynamic interfacial tension were discussed, and a semiempirical equation was established to characterize those effects.