β-Cyclodextrin (β-CD) enhances functional properties by forming inclusion complexes (ICs). This study employed β-CD to form IC with fatty acid ethyl ester (FAEE) for concentrating α-Linolenic acid ethyl ester (ALAEE) from flaxseed oil FAEE, and investigated the interaction mechanisms between β-CD and ALAEE. Using the single-factor method, optimal inclusion conditions yielded an inclusion rate of 61.80 % and increased ALAEE content by 11.77 % (P <
0.05). Antioxidant activity improved by 1.32-fold after concentration (P <
0.05). Differential scanning calorimetry (DSC) indicated successful FAEE inclusion, evidenced by a dehydration peak shift to higher temperatures. Changes in characteristic peaks observed in fourier transform infrared (FTIR) spectroscopy and low-field nuclear magnetic resonance (LF-NMR) confirmed intermolecular interactions in IC. β-CD formed aggregates with FAEE via hydrogen bonding, with ALAEE stablishing a more stable IC due to stronger hydrogen bonding compared to other FAEEs.