Abstract: This paper took Penaeus monodon as the study object to try to optimize fatty acids extraction of marine biological samples by comparing the extracting means and choosing different transfer regents.The results suggested that the method using microwave extraction with n-hexane and methanol (containing 5% HCl) as transfer reagents was the best method.The recovery of this method was higher than 90%.Moreover, the experiment procedure was simplyfied and the extraction time was shortened.Meanwhile, this method had no selective influence on the relative content of individual fatty acids, and could be widely used in the extraction of fatty acids of marine biological samples.