Based on nitrocellulose membrane, we optimized the blocking condition, the volume and concentration of coating antibody. Then, an improved Double Antibody Sandwich Dot Enzyme Linked Immunosorbent Assay (DAS-Dot-ELISA) for Acidovorax avenae subsp. citrulli, the casual agent of bacterial fruit blotch of watermelon, was established. Use adding Disodium Ethylenediamine Tetraacetic Acid (EDTA) in nonfat dry milk treated with high temperature as blocking solution can effectively lower the background. Shaking gently can enhance hybridization and reduce the nonspecific binding. This improved DAS-Dot-ELISA assay detected Acidovorax avenae subsp. citrulli as low as 1.9 × 105 CFU/mL quickly and economically. On the detection of two batches seed sample, the germ-carrying rate with improved DAS-Dot-ELISA was 8.0%, 6.0% which was completely coincident with microwell plate ELISA. On the result of each seed, this improved method was coincided with microwell plate ELISA in 99%, showed a satisfactory prospect and provided a new rapid assay for the detection of Acidovorax avenae subsp. citrulli.