Oil was produced from sugarcane leaves hydrolysate (SLH) in the newly isolated oleaginous yeast Cyberlindnera subsufficiens NG8.2, using a two-stage cultivation method. The SLH contained sugars derived from both the dilute acid pretreatment of leaves and subsequent enzymatic hydrolysis (16.6 g/L of glucose and 15.87 g/L of xylose). The Cyberlindnera subsufficiens NG8.2 produced oil containing 18.73 wt% of palmitoleic acid, with a 0.99-g/L oil yield when grown in the SLH. Removal of phosphate from the SLH by Ca(OH)2 treatment (SLH-P) resulted in an increased oil yield of 1.38 g/L, but the palmitoleic acid content of the oil decreased to 12.45 wt%. Supplementation of the SLH-P with 3.12 mM of Mg2+ increased the palmitoleic acid content in the oil to 15.80 wt% and the oil yield to 1.58 g/L, with a palmitoleic acid yield of 2.09 mg/g sugarcane leaves. Thus, sugarcane leaves are a promising feedstock for palmitoleic acid production using Cyberlindnera subsufficiens NG8.2.