Abstract: In order to develop functional sprouted foods rich in α-linolenic acid (ALA), this study investigated the effects of incubation temperature, salt concentration, and pH on the ALA content during the sprouting process using flaxseed as the raw material. Based on the results of single-factorial experiments, Box-Behnken response surface methodology was used to optimize the sprouting conditions, and the quality and bioactivity of sprouted flaxseed were comprehensively evaluated. The results indicated that the optimal germination time was 48 h, and the optimized process parameters were an incubation temperature of 26 ℃, a salt concentration of 0.5 mg/mL NaCl, and a pH of 6.0, resulting in an ALA content of 20.42 g/100 g. Under these conditions, the activities of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) were significantly enhanced. Meanwhile, the expression of fad2, fad3, fad7, and fad8 genes was significantly upregulated (P<0.05), while the expression of lox gene was downregulated (P<0.05), thereby promoting the synthesis and accumulation of ALA. When flax seeds were sprouted for different periods of time under the optimal sprouting conditions, it was found that the sprouted flax seeds had better nutritional value compared with the unsprouted ones, with the ALA content reaching 24.125 g/100 g (P<0.05), an increase of 13.125 g/100 g. The protein content was increased to 25.75% (P<0.05), the lignan content was increased by 1.29 mg/mL (P<0.05), and the γ-aminobutyric acid (GABA) content was increased by 28.99% (P<0.05), while the cyanogenic glycoside content was reduced by 97.62% (P<0.05). In addition, bioactivity was significantly enhanced (P<0.05), with superoxide dismutase (SOD) activity increasing by 154.25 U/mL (P<0.05). The antioxidant capacity was also significantly improved, with the DPPH and ABTS⁺ free radical scavenging rates increasing by 12.51% and 41.67%, respectively (P<0.05), and the ferrous ion reducing power reaching 0.88 mmol/L (P<0.05). In conclusion, the biosynthesis and accumulation of ALA in flaxseed were significantly improved by optimized germination conditions, and its nutritional quality and bioactivity were enhanced, providing theoretical basis and technical support for the development of healthy foods rich in ALA.