Abstract: Acyl-homoserine lactones ( AHLs) compound is a class of the most important signaling molecule in quorum sensing system of gram-negative bacterium, this paper simulates 5 dominant AHLs ( C₄-HSL, C₆-HSL, C₈-HSL, C₁0-HSL, and 3OC₁2-HSL) generated from gram negative bacteria in freshwater fish, and screens the macrophage strain with sensitive identification of specific AHLs by constructing two macrophage models of Ana-1 and RAW 264.7.The results showed that each of the 5 AHLs could cause the decrease of Ana-1 and RAW 264.7 cell activeness.Through the intracellular Ca ⁽2 +) fluorescence detection, under C₁0-HSL stimulation, the activity of Ana-1 cells showed a certain correlation with intracellular Ca ⁽2 +) , it was speculated that C₁0-HSL could induce the intracellular Ca ⁽2 +) increase in Ana-1 cells, thus trigger the fluorescence reaction of intracellular Ca ⁽2 +) probe. Meanwhile the sensitivity of Ana-1 cells C₁0-HSL was verified by flow cytometry and electron microscopy, which provided the basis and guidance for the construction of ANA-1 cell based fluorescent sensing method for detection of signaling molecule C₁0-HSL in freshwater fish spoilage organisms.