[Objective] Different types, quantities and varying patterns of the AHLs released from pure cultured Pseudomonas fluorescens and Pseudomonas aeruginosa as the standard strains of Pseudomonas spp. were studied in this research. [Methods] AHLs in the pure culture solution were extracted via ethyl acetate and then identified through HPLC-MS-MS. [Results] The results showed that C4-HSL, C6-HSL, C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL, 3-oxo-C14-HSL were the main signaling molecules released from Pseudomonas fluorescens while C4-HSL, C6-HSL, C8-HSL, C10-SL, C12-HSL, C14-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL, 3-oxo-C14-HSL were the corresponding signaling molecules in Pseudomonas aeruginosa. [Conclusion] Quantities of signal molecules from the two strains were changing with time, when the number of colonies at 109?1010, reached the peak of the signal molecule. Also, significant differences of signaling molecules contents released from the 2 strains were found in this research.