Abstract Renewable fuels are major alternatives to conventional fossil fuels. Biomass in the form of agricultural and industrial residues is fast becoming popular among new renewable energy sources. Hydrothermal liquefaction can thermochemically convert biomass residues into bio-oil. This work investigates the hydrothermal liquefaction of microalgae Dunaliella tertiolecta cake under various liquefaction temperatures, holding times, and catalyst dosages. A maximum bio-oil yield of 25.8% is obtained at a reaction temperature of 360 °C and a holding time of 50 min using 5% Na2CO3 as a catalyst. The various physical and chemical characteristics of bio-oil obtained under the most suitable conditions are determined, and a detailed chemical compositional analysis of bio-oil is performed using an elemental analyzer, Fourier transform-infrared spectroscopic analysis (FT-IR), and gas chromatography–mass spectrometry (GC–MS). The bio-oil is composed of fatty acids, fatty acid methyl esters, ketones, and aldehydes. Its empirical formula is CH1.44O0.29N0.05, and its heating value is 30.74 MJ/kg. The bio-oil product is a possible eco-friendly green biofuel and chemical.