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To enhance the stability of lipases, the additive method is attractive because of its high efficiency and simplicity, of which β-cyclodextrin (β-CD), as an additive has attracted attention. In the present study, the effects of β-CD on enzymatic hydrolysis of hemp seed oil by lipases were investigated. The stability, spectroscopy, and reusability of Candida sp.99–125 were compared by the addition of β-CD, respectively. The results showed that Candida sp.99–125 could produce the highest yield of α-linolenic acid (ALA, 18.27%, w/w) in the presence of β-CD after 24h, and the thermal stability of lipase was also improved. The UV spectroscopy showed that the absorbance of lipase decreased with increasing concentrations of β-CD and the fluorescence result was similar. Moreover, the reusability of lipase with β-CD was better than free lipase. This study revealed that β-CD could increase hydrolysis activity and stability of the lipase, thereby improving the hydrolytic process of hemp seed oil by Candida sp.99–125.