Abstract: Aquaculture the rearing of fish and other aquatic animals is the best approach for meeting the increasing demand for aquaculture products worldwide. However, the problem of off-flavor has been documented as a serious setback to aqua-culture. Though there are many off-flavors can result in a fish being objectionable, approximately 80% of the rejected fish have the earthy-musty odor, which is primary caused by the presence of either 2-methylisoborneol (2-MIB) or geosmin. Based on the fact that the detection of earthy-musty odorous compounds in fish mainly relied on the sensory method in Chi-na, the present study established the instrumental method to detect 2-MIB and geosmin by microwave mediated distillation with headspace solid-phase microextraction and gas chromatography-mass spectrum (MWDE-SPME-GC-MS). Through in-vestigating the effects of the key parameters, microwave processing time and carrier gas flow rate, in microwave mediated distillation on extracts,it is concluded that 6 min of microwave time and 70 mL/min nitrogen flow were the optimized con-dition on which earthy-musty odorous compounds could be completely distilled from fish flesh. Then the earthy-musty odor-ous compounds were absorbed in the fiber under HS-SPME, and were finally desorbed at 250℃ and determined by GC-MS. The limits of detection for 2-MIB and geosmin in fish were both 0.1 μg/kg. There were significant linear correlation in the range of 1-20 μg/kg for the two earthy-musty odorous compounds, with calibration coefficients at 0.987 and 0.995, respectively. Therefore, trace levels of earthy-musty odorous compounds at ppb in fish could be quantified by this method with credible results.