This work addresses the conversion of wet microalgae to levulinic acid (LA) using a one-pot reaction system. Utilizing moisture in microalgae forms a biphasic system with an organic solvent of 1, 2-dichloroethane (DCE) is formed. This system enhances the LA yield by making an acidic environment through the decomposition of DCE in a small quantity and the recovery of products in each aqueous and organic phase. With lipid-richNannochloropsis gaditanaand carbohydrate-richChlorellaspecies, the effects of reaction variables of temperature, water content, and DCE dosage on the LA production were investigated. The LA yield was 30.13 wt% and 28.15 wt% based on the mass of total hexoses (43-47 wt% of convertible hexoses) for the two types of microalgae at 160 degrees C, while the yield of free fatty acids reached 90.13 w/w% at 180 degrees C based on the esterifiable lipid. This biphasic system facilitates the forward reaction and the product recovery for concurrent reaction and separation.