A method was developed using a padlock probe (PLP) and dot-blot hybridization for detecting Acidovorax citrulli in cucurbit seed. The PLP was designed based on the 16S-23S internal transcribed spacer ribosomal DNA sequence from A. citrulli. The detection threshold for the PLP assay was 100 fg of genomic DNA, and A. citrulli was detected in 100% of artificially infested seedlots with 0.1% infestation or greater. In addition, using the PLP assay, 4 of 8 melon seedlots collected from Xinjang province and 15 of 47 watermelon seedlots collected from Ningxia province were positive for A. citrulli. In contrast, a conventional polymerase chain reaction (PCR) assay that relied on primers WFB1 and WFB2 facilitated A. citrulli detection in 1 of 8 and 5 of 47 seedlots from Xinjiang and Ningxia provinces, respectively. These data indicate that the PLP and dot-blot hybridization technique was more effective than conventional PCR for seed health testing.