Background and Objectives: As a green fuel and environmentally friendly energy, biodiesel has recently attracted much attention and efforts are ongoing to optimizing biodiesel production from microalgae’s. The aim of this study was to determine the appropriate method of dewatering and drying biomass and selecting a suitable organic solvent for extraction lipids from biomass. Materials and Methods: After culturing Nannochloropsis Oculata in Gillard F/2 medium and reaching at the end of the stationary growth phase, algal biomass was separated from aqueous by centrifuge and drying in three methods: fore, air-dried and lyophilized. Lipid extractions of each sample was performed using soxhlet apparatus and three solvents including diethyl ether, n-hexane and n-pentane. At each stage, the quantity and quality of the extracted lipids was determined by gas chromatography. Results: In all three drying methods, palmitic acid and palmitoleic acid were significantly the main fatty acid composition of microalgae. The fatty acid composition of palmitic acid extracted by diethyl ether was significantly more than the other two solvents. Maximum production of triglyceride was observed in air dried and lyophilized (using diethyl ether solvent) microalgae as 75.03 and 76.72 % of fatty acid respectively. Conclusion: The use of lyophilized method for dewatering and drying of biomass and diethyl ether as solvent for the extraction of lipids from biomass yielded more compared with other methods studied in this paper and would be more efficient in research works related to the production of biodiesel from microalgae’s lipid.