Abstract: A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 105 colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.