A bilayer microcapsule (BM) was prepared by layer self-assembly method with lemon seeds oil (LSO) as the core material, and yeast cell walls (YCW) and esterified starch as the wall materials to examine its effect on the preservation of beef jerky (BJ). The bilayer structure significantly improved the oil load capacity (193.00 mg/g), thermal stability, and moisture adsorption (up to 2.3 g/10 g) of BM compared to a single-layer structure (P < 0.05). The release mechanism followed Fick's law of diffusion. After 300 d of storage, BM significantly inhibited the increase in thiobarbituric acid (0.84 mg MDA/kg) and volatile salt nitrogen (10.32 mg/100 g) content and growth of microorganisms (4.82 lg CFU/g) compared with conventional desiccants (P < 0.05). Meanwhile, BM maintained high sensory scores (odor, color, overall acceptability) including that it can replace traditional desiccants to extend the shelf life of BJ products.