Abstract Developing technologies for the production of biofuel from renewable resources is a field of interest for many researchers. Lipid extraction could be an important step in the microalgae biodiesel production process. Factors affecting intracellular lipid extraction from Chlorella sp. cultivated in outdoor raceway ponds were investigated; an optimized procedure for extraction of total and non-polar lipids using eco-friendly solvent combination of ethyl acetate and methanol was proposed. The effects of solvent, and extraction variables (temperature, time, ratio of solvent and biomass, ratio of ethyl acetate and methanol) on total lipid content, and lipid class were examined via single-factor experiments coupled with response surface methodology (RSM) using Box−Behnken design (BBD). The results revealed that the maximum lipid extraction yield was 18.1% obtained after extraction 120 min, extraction temperature 60 °C and M/EA ratio was 2:1. Fatty acid profiles of lipid were determined; palmitic acid (C16:0), palmitoleic (C16:1) oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) are the most abundant fatty acids, indicating the great capacity of lipid extraction from microalgae for biodiesel production.