Background: The extraction of lipids from microalgae requires a pretreatment process to break the cell wall and subsequent extraction processes to obtain the lipids for biofuels production. The multistep operation tends to incur high costs and are energy intensive due to longer process operations. This research work applies the combination of radicals from hydrogen peroxide with an organic solvent as a chemical pretreatment method for disrupting the cell wall of microalgae and simultaneously extracting lipids from the biomass in a one-step biphasic solution. Result: Several parameters which can affect the biphasic system were analyzed: contact time, volume of solvent, volume ratio, type of organic solvent, biomass amount and concentration of solvents, to extract the highest amount of lipids from microalgae. The results were optimized and up to 83.5 of lipid recovery yield and 94.6 of enhancement was successfully achieved. The results obtain from GC-FID were similar to the analysis of triglyceride lipid standard. Conclusion: The profound hybrid biphasic system shows great potential to radically disrupt the cell wall of microalgae and instantaneously extract lipids in a single-step approach. The lipids extracted were tested to for its comparability to biodiesel performance. © 2019 The Author(s).