In situ transesterification (direct conversion) of microalgal cells is a promising method to produce biodiesel from microalgae because it integrates the oil extraction and conversion process in one step. Not only biodiesel but also a few biochemicals can be produced through this process because both lipids and carbohydrates are converted under acidic conditions. Levulinic acid ester (levulinate) is one of the byproducts of in situ transesterification, which can be used as an additive in fuels or fragrances. This study investigated the effect of cell composition and reactive variables on the productivity of levulinic acid ester. The cell compositions of microalgal strains were compared between Nannochloropsis and Chlorella, and more levulinate was produced from carbohydrate-rich Chlorella cells. Both reaction temperature and acid concentration highly affected the levulinate yield, whereas the type of alcohols did not have much influence on the yield. Consequently, more than 40 mol% glucose inside the cell was converted to levulinate with a 15 v% sulfuric acid concentration at 130 °C.