Abstract: To investigate the inhibitory effects of bio-derived mixed fatty alcohols obtained through temperature gradient fractionation on tobacco axillary bud growth and develop an efficient, long-lasting, and safe bio-bud inhibitor, this study obtained three bio-derived mixed fatty alcohol fractions (low-temperature alcohol, LMA; medium-temperature alcohol, MMA; high-temperature alcohol, HMA) via temperature gradient fractionation. The HMA fraction was further modified through hydroxyl esterification to obtain fatty acid methyl ester (HME). Gel permeation chromatography and Fourier transform infrared spectroscopy analyses revealed that the molar mass increased with rising fraction temperature, with LMA, MMA, and HMA exhibiting molar masses of 110-150 g/mol, 200-280 g/mol, and 300-380 g/mol, respectively. Four formulations prepared through dosage formulating processing exhibited stable storage properties, with their dilution solutions forming milky white homogeneous emulsions with negative charges, contact angles below 50°, and surface tensions ≤30 mN/m, showing excellent wettability on the wax layer of tobacco axillary buds. Inhibitory effects showed that LMA exhibited outstanding rapid efficacy with a 95.06% bud inhibition rate at 15 days, but with poor persistence. MMA displayed rapid and sustained effects, achieving 96.00% inhibition rate at 30 days and a maximum inhibition efficiency of 97.46%. HMA showed poor permeability and difficulty in formulating high-concentration products, with inhibitory efficiency comparable to fluoroacetamide. The inhibitory effect of HME significantly decreased to 26.80%, suggesting that hydroxyl groups are the key active functional moieties. In summary, the inhibitory agent prepared from medium-temperature fraction mixed fatty alcohols demonstrated optimal rapid and sustained efficacy, achieving the best performance.