Abstract: Brassinosteroid is a steroidal phytohormone involved in responses to stress, and CYP734A1 gene is a crucial gene on the metabolic pathway of brassinosteroids. To investigate the function of the CYP734A1 gene in response of Nicotiana tabacum to abiotic stress, this study used the Nicotiana tabacum variety K326 as the material, the NtCYP734A1 gene was cloned, and the physicochemical properties and evolutionary relationship of NtCYP734A1 protein were analysed by using bioinformatics technology. The expression pattern of NtCYP734A1 gene in various tissues and different abiotic stress treatments was analyzed through real-time fluorescence quantification. The results showed that NtCYP734A1 gene was 1 638 base pairs long and encoded for 545 amino acids. Subcellular localization predicted that NtCYP734A1 protein existed in the endoplasmic reticulum. Analysis using qRT-PCR revealed that Nicotiana tabacum NtCYP734A1 was expressed in different tissues, with the highest expression observed in the leaf and the lowest in the stem. The gene’s expression was significantly changed in various tissues under abiotic stress treatments. The expression of NtCYP734A1 in leaves decreased under four types of stress: drought, salt, high temperature, and low temperature.