Biodiesel (BDF) production from microalgae is a promising technology because of rapid growth rate compared with other biomass plants and high productivity of bio-oil. In this study, we evaluated production cost and net energy production of a large-scale BDF production system, which combines hydrothermal liquefaction for bio-oil extraction and supercritical methanol transesterification, from microalgae (process 1). The unit cost of BDF production in process 1 is 5.5×10^2 yen (L-BDF)^<-1>, which is lowest among the processes. It was found construction cost of culturing pond must be significantly reduced and that nutrients must be recycled for commercialization.