To screen lactic acid bacteria （LAB） with inhibitory activity of quorum sensing （QS） and biofilm formation of Vibrio harveyi from fish intestine， five strains LAB with inhibitory activity against QS of V. harveyi were screened from 30 strains LAB by the Oxford cup agar well diffusion method. Among them， strain LY3-1 isolated from Cyprinus carpio showed strong anti-QS activity and the inhibition diameter was 14.21 mm. The degradation rate of crude extracts from strain LY3-1 on acylated homoserine lactones （AHLs） signal molecular was 41.7%， and its biofilm inhibition rate was 69.8% at the mass concentration of 8.0 mg/mL. Moreover， it could completely degrade AHLs signal molecular， and its biofilm inhibition rate was 90.6% at the mass concentration of 16.0 mg/mL. The inhibitory activity of the crude extracts treated by proteases on quorum sensing of V. harveyi was lossed， indicating that the crude extracts had protein properties. After treating at 40-121 ℃ for 30 min， there was no significant difference in quorum sensing inhibitory activity of crude extracts （P>0.05）， indicating that the crude extracts had heat resistance； and the crude extracts of strain LY3-1 with quorum-sensing inhibitory activity at pH 3.5-7.0. The results of light and scanning electron microscopy revealed that the crude extracts of strain LY3-1 inhibited the formation of V. harveyi biofilms. The strain LY3-1 was identified as Lactococcus lactis by physiological and biochemical characteristics and 16S rRNA gene sequencing. The study indicated that Lc. lactis could be used as a novel quorum sensing inhibitor of gram-negative bacteria. It will provide fundamental basis for the development of QS inhibitor for gram-negative bacteria quorum sensing.