A high-throughput screening method for bacterial strains capable of generating quorum sensing signal molecules comprises the following steps: step 1, preparing a reporter plate which is a solid microbiological medium with distributed reporter strains; step 2, separating to-be measured strain to obtain single colonies of the to-be measured strain; step 3, transferring the to-be measured strain: photocopying the single colonies of the to-be measured strain to the reporter plate described in the step 1 by using a film covering method; and step 4, obtaining the strain capable of expressing the signal molecules: culturing the reporter plate described in the step 3 until the expression signal is detectable, wherein the bacterial strain, which is in the single colonies of the to-be measured strain and is corresponding to the position of displaying the expression signal in the reporter plate, is the bacterial strain capable of expressing the signal molecules. The method is capable of screening 40-60 single colonies in one time and further improving screening efficiency when the screening platform is enlarged, and the screening obtained results are reliable, and the generation rates of false positive and false negative are low.