[Objective] To detect a novel signaling molecule of quorum sensing, diketopiperazines (DKPs), in spoilage bacteria of aquatic products, the simple and sensitive technique of GC-MS method was developed for the quantitative analysis. [Methods] By optimizing the conditions of gas chromatography and mass spectrometry, media and extracting solvent, the quantitative measure of diketopiperazines (DKPs) was established. The characteristic ions of four kinds of DKPs standards, cyclo-(L-Pro-L-Gly), cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe), was ensured. The DKPs activity in two spoilage bacterial of aquatic product, Pseudomonas fluorescens and Shewanella baltica, were detected. [Results] Four DKPs had good linear at range of 1?200 mg/L. The limits of detection were 0.06, 0.10, 0.06 and 0.04 mg/L, respectively. The limits of quantification were 0.16, 0.18, 0.14 and 0.12 mg/L, respectively. The recovery of four DKPs in the medium was among 51.8%?88.5% with the RSDs of 1.4%?8.3%. The higher level of DKPs was detected with chloroform served as extracting solvent and LB used as medium. Two kinds of DKPs, including cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), were found in P. fluorescens and S. baltica. The level of DKPs in two spoilage bacteria increased with the bacterial growth, and arrived to the highest activity for 12 h of culture. [Conclusion] The technique of GC-MS method developed could analyze quantitatively the levels of four kinds DKPs in spoilage bacteria, with high precision and accuracy.