In microfluidic-based liposome preparation, ethanol affects liposome size and the structural changes during liposome formation and dialysis. We reported the effects of the flow conditions and microchannel structure on the liposome size. Generally, large liposomes form at a slow ethanol dilution rate, whereas small liposomes form at a rapid ethanol dilution rate. Additionally, the liposome suspension prepared using the microfluidic device contains ethanol, and residual ethanol may induce fusion and structural changes within the liposomes. Understanding the effects of time-dependent changes in ethanol concentration on liposome formation and ethanol removal processes, such as dialysis, enables the development of novel microfluidic devices for more precise size and structural control, including lamellarity and higher drug loading efficiency. Transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) spectroscopy, and small-angle X-ray scattering (SAXS) are generally used to determine the liposome structure under static conditions, but they lack the temporal resolution to determine the changes in the liposome structure. The measurement of structural changes in liposomes with time-dependent changes in ethanol concentration, in particular, is a major challenge in the fields of nanomaterial science, structural biology, and analytical chemistry. To understand the effects of time-dependent changes in ethanol concentration on the structural changes in liposomes, time-resolved SAXS using a rapid stopped-flow mixing technique or microfluidic devices is a suitable measurement method.