Powdery mildew poses a significant threat to watermelon production. The development of disease-resistant varieties through gene editing represents a major focus in current breeding research. In this study, we identified an MLO family gene in watermelon, denoted by ClMLO5b, which is phylogenetically closely related to cucumber CsaMLO8 and melon CmMLO5. Homology modeling revealed high conservation of the three-dimensional protein structures among these orthologs. Expression analysis demonstrated that ClMLO5b is significantly up-regulated upon powdery mildew infection, and the protein localizes to the plasma membrane. To validate its function, we first employed an Agrobacterium rhizogenes-mediated hairy root transformation system to rapidly verify the editing efficiency of two CRISPR/Cas9 targets designed for ClMLO5b. Subsequently, stable transgenic watermelon plants were generated via Agrobacterium tumefaciens-mediated transformation, and a mutant line with homozygous substitutions at target site 2 was obtained. Disease resistance assays showed that, compared to wild-type plants, the Clmlo5b exhibited strongly inhibited mycelial growth, significantly reduced disease severity, and a substantial decrease in spore production after inoculation with powdery mildew. Our findings confirm that ClMLO5b is a key susceptibility gene in watermelon and provide both a promising genetic target and valuable breeding material for developing powdery mildew-resistant watermelon varieties.