The rapid and sensitive detection of Alicyclobacillus acidoterrestris (AA) has become very important due to the frequent occurrence of fruit juice spoilage by AA. In the present study, using guaiacol, both as the metabolic product of AA related to its concentration and as a green colorimetric substrate of G-quadruplex DNAzyme, a novel G-quadruplex DNAzyme-based colorimetric method for a rapid detection of AA has been developed for the first time. Under optimal conditions, AA has been successfully detected in the concentration range of 10(2)-10(5) cfu mL(-1) with a detection limit of 85 cfu mL(-1). The recoveries ranging from 71.8% to 115.7% with relative standard deviation from 1.2% to 6.6% in spiked apple and orange juice samples were obtained. Results demonstrate that the sensitivity and precision of the developed method is comparable with most other analytical methods and is prominently rapid than them. We believe that the work provides a novel and effective approach and is beneficial for monitoring and reducing the risk of AA contaminations during the process of fruit juice production.