Abstract: N-acyl-homoserine lactones(AHLs)are typical quorum sensing signal molecules and regulate the expression of many physiological characteristic in V.parahaemolyticus. At present,there is a lack of accurate detection methods for AHLs in biofilms. Based on the existing conditions of HPLC-MS/MS,detection methods for AHLs could be optimized during the biofilm formation of V. parahaemolyticus. It was identified that absolute methanol:0.1% ammonium acetate water as the mobile phase separation could help to obtain better results. If the collision energy was 10~15 eV,a higher proportion of characteristic fragment ions(m/z)102.1 and 74.1 could be formed for each signal molecule. Good linear relationship was obtained in the wide range of 0~20 μg/L(R²>0.995). This method could help to detect three different signal molecules,including C4-HSL,3-oxo-C6-HSL and 3-oxo-C14-HSL,which could be formed in the formation process of biofilms of V.parahaemolyticus. Among these signal molecules,the amount of C4-HSL was higher than others(>91.67%).In the early stage of biofilm formation(0.5~3 d),the concentration of AHLs increased slowly and was positively relate to the biofilm formation. In the discursive stage(4~5 d),the concentration of signa molecules increased rapidly. Furthermore,a negative correlation could be found significouldtly between the concentration of signal molecules and biofilm formation. The optimization of this method could help to effectively detect the AHLs of biofilm and provided support for further study of regulative relations between biofilm and the quorum-sensing as well.