The volatile oil of Gracilaria lemaneiformis was extracted by direct steam distillation and solvent extraction-steam distillation, meanwhile gas chromatography-mass spectrometry (GC-MS) and antibacterial & insecticidal activity were conducted. The GC-MS showed that compared with direct steam distillation extraction, in solvent extraction-steam distillation extraction the relative content of aldehyde compounds which has lower odor threshold is 0.20%, meanwhile the relative content of carboxylic acid and ester is 21.54% in solvent extraction-steam and distillation extraction. The antibacterial activity of volatile oil on 5 kinds of pathogens （Escherichia coli O157, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, Vibrio parahemolyticus） is moderate, and the median lethal dose LD₅₀ of volatile oil compounds in G. Lemaneiformis with solvent extraction-steam distillation and direct steam distillation broth were 0.126 and 0.162 mg/mL respectively.