<p>Onion (<i>Allium cepa</i> L.) is one of the most economically important crops worldwide. During onion cultivation, various pathogens cause significant losses, with <i>Botrytis squamosa</i>, the causal agent of leaf blight, being one of the notable pathogens affecting onion crops. Although chemical and physical control methods are widely used to control <i>B. squamosa</i>, these are labor-intensive and can cause environmental pollution. Therefore, further studies are required to develop breeding strategies for leaf blight resistance. However, as a biennial plant with a large genome size (&gt; 16 Gb), onions have a long breeding period. Additionally, at present, research on the functional analysis of genes related to resistance to leaf blight in onions is scarce. Therefore, in this study, <i>allene oxide synthase</i> (<i>AOS</i>) obtained from transcriptome analysis of leaf blight-resistant and -susceptible onion lines were evaluated for resistance to <i>B. squamosa</i>. The expression levels of <i>A</i>. <i>cepa AOS</i> (<i>AcAOS</i>) were analyzed after the inoculation of both resistant and susceptible onion lines. Then, <i>AcAOS</i> was isolated from the resistant onion and <i>AcAOS</i> overexpression and downregulation (RNA<i>i</i>) vectors were constructed to analyze <i>AcAOS</i> gene function. Since an onion transformation method has yet to be established, <i>Nicotiana tabacum</i> was transformed with these vectors. The resistance degree of transgenic lines was confirmed after inoculation, and the expression levels of <i>AOS</i> and <i>allene oxide cyclase</i> (<i>AOC</i>), downstream genes in the α-linolenic metabolism pathway, were analyzed. The results indicated that <i>AcAOS</i> is related to resistance to leaf blight in onions.</p>