Tobacco bacterial wilt is a destructive soil-borne vascular disease in tobacco production, and it cannot be effectively controlled by bactericides. SYP-14288 is a typical uncoupler with a broad spectrum, but the action site of SYP-14288 is unknown. The objective of this study was to assess resistance risk and clarify resistance mechanism of Ralstonia solanacearum to SYP-14288. In this study, the inhibition spectrum of SYP-14288 showed that it had a good inhibitory effect on 10 plant pathogenic bacteria. The sensitive baseline of tobacco bacterial wilt to SYP-14288 was established, and the average EC₅₀ values were 0.0378 µg/ml. Sixteen resistant mutants were obtained using SYP-14288-amended media method, which can be divided into low resistance, medium resistance and high resistance levels. Based on the survival suitability of sensitive isolates and resistance mutants, the resistance risk of tobacco bacterial wilt to SYP-14288 could be moderate. Through the joint analysis of transcriptome and proteome, the key metabolic pathways and main differential genes were cleared. The drug efflux mediated by ABC transporter was one of the main reasons for the resistance of tobacco bacterial wilt to SYP-14288. This study could provide a technical support for the rational use of SYP-14288 and delaying the development of resistance in fields.