Objective: To obtain a promising production technique of conjugated linoleic acid (CLA). Methods: The alkali isomerization was used to prepare CLA from peanut oil. The relative content of linoleic acid in peanut oil was determined, and the effects of reaction time, reaction temperature, ratio of alcohol to oil and KOH dosage on the conversion of CLA were investigated. The preparation conditions were optimized by Box-Behnken test. Results: The peanut oil was rich in linoleic acid with a relative content of 27.85%. Therefore, peanut oil was a good raw material for the preparation of CLA. For the conversion of CLA, reaction temperature, reaction time and KOH dosage were the most significant factors (P＜0.01), while ratio of propylene glycol to oil was the significant factor (P＜0.05). There existed interaction between reaction time and ratio of propylene glycol to oil. The best conditions for preparation of CLA by alkali isomerization of peanut oil were as followed: the reaction temperature of 200 ℃, the reaction time of 2.6 h, the ratio of propylene glycol to oil of 22 to 1, the KOH dosage of 7% (mass fraction). Under determined conditions, the conversion of CLA was 89.81%. Conclusion: It is workable to prepare CLA by alkali isomerization of peanut oil, and the conversion of CLA is high under the optimal conditions.