Prebiotic membranes are one of the essential elements of the origin of life because they build compartments to keep genetic materials and metabolic machinery safe. Since modern cell membranes are made up of ethanolamine-based phospholipids, prebiotic membrane formation with ethanolamine-based amphiphiles and phosphates might act as a bridge between the prebiotic and contemporary eras. Here, we report the prebiotic synthesis of O-lauroyl ethanolamine (OLEA), O-lauroyl methyl ethanolamine (OLMEA), and O-lauroyl dimethylethanolamine (OLDMEA) under wet-dry cycles. Turbidimetric, NMR, DLS, fluorescence, microscopy, and glucose encapsulation studies highlighted that OLEA-ATP and OLMEA-ATP form protocellular membranes in a 3:1 ratio, where ATP acts as a template. OLDMEA with a dimethyl group did not form any membrane in the presence of ATP. ADP can also template OLEA to form vesicles in a 2:1 ratio, but the ADP-templated vesicles were smaller. This suggests the critical role of the phosphate backbone in controlling the curvature of supramolecular assembly. The mechanisms of hierarchical assembly and transient dissipative assembly are discussed based on templated-complex formation via electrostatic, hydrophobic, and H-bonding interactions. Our results suggest that N-methylethanolamine-based amphiphiles could be used to form prebiotic vesicles, but the superior H-bonding ability of the ethanolamine moiety likely provides an evolutionary advantage for stable protocell formation during the fluctuating environments of early earth.