The quality problems of acidic juices such as nutritional loss， flavor deterioration and precipitation caused by the pollution of acidophilic and thermophilic Alicyclobacillus acidoterristris have become the bottleneck restricting the processing and export trade of fruits and vegetables. In this paper， the antibacterial activity of inosine on growth of A. acidoterristris and its influence on the quality of orange juice were studied by determination of optical density， measurement of biofilm formation， scanning electron microscopy (SEM) detection， plate colony count， physicochemical properties and sensory quality identification of fruit juice. The results showed that inosine with different concentrations could effectively inhibit the growth and reproduction of the vegetative cells of A. acidoterristris under acidic conditions， and had no significant effect on the physicochemical properties such as acidity， sugar content， color， taste and sensory quality of fresh orange juice. The addition of inosine as low as 5 mmol/L could reduce the biofilm formation ability of A. acidoterristris， damage the cell wall and cell membrane， and lead to cell lysis and death. When the initial contaminated bacteria content is up to 105 CFU/mL， the total number of A. acidoterristris can still be reduced by one order of magnitude， which control the number of vegetative bacteria so that it can not produce guaiacol that reaches the olfactory threshold. It is expected to be a new and efficient preservative for inhibiting the pollution of A. acidoterristris in acidic juice production.