Abstract: To select the optimum mobile phase and injection, the influence of solvent on sensitivity of nitrofuran metabolites (AMOZ, SEM, AHD and AOZ) was investigated by liquid chromatography tandem mass spectrometry (LC-MS/MS). The highest intensity of AHD was obtained in Q1 scan with negative mode. However, the satisfactory sensitivities of four nitrofuran metabolites could be gained in positive mode while the compounds were dissolved in methanol that contained 0.1% aqueous ammonia. Gradient elution for LC separation was performed by using methanol and 0.1% aqueous ammonia in water solution, and the injection was also added with 0.1% aqueous ammonia. The optimized method is suitable for determining of four nitrofuran metabolites in multiple matrices with high sensitivity, less interference and excellent accuracy.