Abstract: A novel hydrothermal catalytic liquefaction process for Chlorella pyrenoidosa was studied in the paper to explore the new resources of bio-fuel. The effects of catalytic liquefaction temperature, reaction time and catalyst on the yield of bio-fuel were investigated, and the reaction conditions were optimized via orthogonal optimization method. The chemical groups and components of final bio-fuel were characterized by Fourier transform infrared spectrometry (FTIR) and gas chromatography/mass spectrometry (GC-MS). The results showed that the optimal reaction was taken place at 300°C for 20 minutes as Ce/HZSM-5 was selected as a catalyst with 5% mass fraction and the volume ratio of Chlorella pyrenoidosa to solvent was 1:10. The maximum liquefaction yield reached to 39.87% and the heating value of final bio-fuel was up to 26.09 MJ/kg. The main components of bio-fuel from Chlorella pyrenoidosa contained alcohol, ester derivatives and some hydrocarbons. Compared with other lignocellulosic bio-fuels, the prepared bio-fuel can be regarded as an alternative diesel fuel because of their similar chemical components and higher heat value. The research is helpful for the preparation of bio-fuel from microalgaes.