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Paeonia ostii is an authorized novel vegetable oil crop due to its seeds rich in unsaturated fatty acids (UFAs) especially α-linolenic acid (ALA), which overweight the current available edible oil. However, little is known on the regulation mechanism of UFAs biosynthesis during its seed development. Here, we used transcriptome and proteome data combining phytochemistry means to uncover the relationship between abscisic acid (ABA) signaling and UFAs biosynthesis during P. ostii seed development. Based on transcriptome and proteome analysis, two desaturases of omega-6 and omega-3 fatty acid, named as PoFAD2 and PoFAD3 responsible for ALA biosynthesis were identified. Then, an ABSCISIC ACID-INSENSITIVE 5 (ABI5) proteins was identified as an upstream transcriptional factor, which activated the expression of PoFAD3 instead of PoFAD2. Moreover, silencing of PoABI5 repressed the response of PoFAD3 to ABA. This study provides the first view on the connection between the function of ABA signaling factors and ALA biosynthesis in the P. ostii seed, which lays the foundation for studies on the regulatory mechanism of ABA signaling involved in the UFAs synthesis during seeds development, meanwhile, it will shed light on manipulation of ALA content for satisfying human demands on high quality of edible oil or healthy supplement.