Extended-synaptotagmins (E-Syts) are proteins located on the endoplasmic reticulum (ER) that tether the ER to the plasma membrane (PM) and regulate their lipid homeostasis via its lipid transfer module, the synaptotagmin-like mitochondrial lipid–binding protein (SMP) domain. Here, we describe in vitro DNA nanostructure–assisted lipid transfer assays investigating how the SMP domain transports lipids between membranes and associates with the membranes to extract and release lipids. The lipid transfer signal was detected through fluorescence resonance energy transfer (FRET). This method overcomes the limitations of commonly used lipid transfer assays in accurately controlling inter-liposome distance and liposome size, enabling us to further understand the details involved in the process of SMP domain–mediated lipid transfer. Similar platforms can be extended to studying the lipid transfer distance and membrane curvature sensitivity of other lipid transfer proteins.