α-Linolenic acid (ALA) is an important essential omega-3 (ω-3) polyunsaturated fatty acid for the maintenance of human health. Although ALA has traditionally been obtained from plant sources, microbial fermentation has emerged as a promising alternative for its sustainable and cost-effective production. However, most of the present approaches rely on genetically modified organisms, which present regulatory and consumer-acceptance concerns. In this study, we aimed to develop a high-ALA-producing strain of Aspergillus oryzae, a Generally Recognized As Safe (GRAS) microorganism widely used in food production in Japan, through self-cloning, a form of genetic engineering that utilizes only the host’s own DNA. To achieve this, an endogenous ω-3 desaturase gene (fad3), which catalyzes the conversion of linoleic acid to ALA, was identified via BLASTP analysis. Subsequently, a multicopy A. oryzae strain (Aofad3-MC) overexpressing fad3 was constructed. This strain increased ALA production, with ALA comprising 30.7% of the total lipids. Furthermore, k-mer analysis confirmed the absence of foreign vector sequences, verifying that Aofad3-MC was constructed through self-cloning. In addition to the identification of the A. oryzae ω-3 desaturase gene, this study provides a microbial platform for the sustainable production of ALA, with potential applications across the food, feed, and related industries.