In this paper, bioreporter strain Chromobacterium Violaceum 026 (CV026) was used to detect the inhibitory effect of quorum sensing inhibition activity of paeonol. Then anti-quorum sensing and anti-biofilms efficacy of paeonol against Serratia grimessi were further studied. The results indicated that peaonol could inhibit the violacein production secreted from CV026 when the bacteria was the sub-minimum inhibitory concentration (sub-MIC). When peaonol mass concentration was 500 μg/mL, the yield of violacein decreased by 57.95%. The results of optical microscope analysis showed that paeonol could hinder the biofilm formation of Serratia grimessi. The scanning microscope results also manifested the biofilm inhibition of paeonol and further showed a rupture of biofilm caused by paeonol. Furthermore, the expression of virulence factors, such as swarming activity, protease production and extracellular polysaccharide production, had been suppressed after treating with paeonol. With the increase of paeonol, the suppressed effect was shown as the mass concentration-dependently. These results demonstrated that the paeonol had an effective inhibitory activity and could be developed as a potential quorum sensing inhibitor (QSI).