Niosomes are synthetic membrane vesicles formed by self-assembly of nonionic surfactant, often in a mixture with cholesterol and dicetyl phosphate. Because of their inner aqueous core and bilayer membrane shell, niosomes are commonly used as carriers of treatment agents for pharmaceutical and cosmetic applications or contrast agents for clinical imaging applications. In those applications, niosomes are considered as a more economical and stable alternative to their biological counterpart (i.e., liposomes). However, conventional bulk method of niosome preparation requires bulk mixing of two liquid phases, which is time-consuming and not well-controlled. Such mixing conditions often lead to large niosomes with high polydispersity in size and thus affect the consistency of niosome dosage or imaging quality. In this study, we present a new method of niosome self-assembly by microfluidic hydrodynamic focusing to improve on the size and size distributions of niosomes. By taking advantage of the rapid and controlled mixing of two miscible fluids (i.e., alcohol and water) in microchannels, we were able to obtain in seconds nanoscaled niosomes with approximately 40% narrower size distributions compared to the bulk method. We further investigated different parameters that might affect on-chip assembly of niosomes, such as (1) conditions for the microfluidic mixing, (2) chemical structures of the surfactant used (i.e., sorbitan esters Span 20, Span 60, and Span 80), and (3) device materials for the microchannel fabrication. This work suggests that microfluidics may facilitate the development and optimization of biomimetic colloidal systems for nanomedicine applications.