Microalgae are recognized as a third generation feedstock for biofuel production due to its rapid growth rate and lignin-free characteristic. In this study, the lipid extracted microalgae biomass residues was used as the material to produce isoprene, α-pinene and β-pinene with the engineered E. coli strain. We adopted an optimal sulfuric acid hydrolysis method to convert holocellulose into fermentable sugar efficiently (6.37 g/L) and explored a novel detoxification strategy (phosphoric acid/calcium hydroxide) to remove inhibitors notably. 55.32 % acetic acid, 99.19 % furfural and 98.22 % 5-HMF were cut down with the phosphoric acid/calcium hydroxide method, and the fermentation concentration of isoprene (223.23 mg/L), α-pinene (382.21 μg/L) and β-pinene (17.4 mg/L) using the detoxified hydrolysate as the carbon source account for approximately 86.02 %, 90.16 % and 88.32 % of those produced by the engineered E. coli strain fermented on pure glucose, respectively.