Lipids from oleaginous microorganisms, including oleaginous yeasts, are recognized as feedstock for biodiesel production. A production process development of these organisms is necessary to bring lipid feedstock production up to the industrial scale. This study aimed to enhance lipid production of low-cost substrates, namely sugarcane top and biodiesel-derived crude glycerol, by using a two-stage cultivation process with Rhodosporidiobolus fluvialis DMKU-SP314. In the first stage, sugarcane top hydrolysate was used for cell propagation, and in the second stage, cells were suspended in a crude glycerol solution for lipid production. Optimization for high cell mass production in the first stage, and for high lipid production in the second stage, were performed separately using a one-factor-at-a-time methodology together with response surface methodology. Under optimum conditions in the first stage (sugarcane top hydrolysate broth containing; 43.18 g/L total reducing sugars, 2.58 g/L soy bean powder, 0.94 g/L (NH4)2SO4, 0.39 g/L KH2PO4 and 2.5 g/L MgSO4 7H2O, pH 6, 200 rpm, 28 °C and 48 h) and second stage (81.54 g/L crude glycerol, pH 5, 180 rpm, 27 °C and 196 h), a high lipid concentration of 15.85 g/L, a high cell mass of 21.07 g/L and a high lipid content of 73.04% dry cell mass were obtained.