The increasing demand for energy has resulted in the current worldwide energy crisis. The uncontrolled use of fossil fuels has resulted in severe negative impacts on human health and the environment. Therefore, an alternative, renewable natural resource is required to fulfill the world’s energy needs. Indonesia has a potential alternative energy source: microalgae-based biofuel is renewable and can be further developed. The aim of this study was to determine the productivity of microalgae biomass, the effect of temperature and time on the transesterification reaction, content and composition of biodiesel (FAME). The general processes were cultivation using bubble column photobioreactors, harvesting, lipid extraction from the biomass, and biodiesel synthesis using transesterification. The cultivation of 7.5 L Synechococcus HS-9 resulted in 5.3 g of microalgae wet biomass and 3.32 g/L of dry biomass. The synthesis of biodiesel using transesterification was influenced by both temperature and reaction time. Here, a 5.5% biodiesel yield was obtained at 55 °C in 60 min. The highest biodiesel yield was 4.25% in 30 min at 65 °C. The FAME contents of Synechococcus HS-9 microalgae biodiesel were 3.16%, 18.96%, and 77.87% monounsaturated fatty acids, polyunsaturated fatty acids, and saturated fatty acids, respectively.