Abstract:[Background] This project is based on the methods of detection and identification of Alicyclobacillus in our laboratory. We hope to establish a good economic value and practical value, more convenient, fast, accurate, specific and sensitive detection methods. [Objective] In order to detect and identify Alicyclibacillus rapidly in raw materials or finished products in fruit juice production enterprises. [Methods] We immunized BALB/c mice with A. acidoterrestris (ATCC49025) and established indirect ELISA to screen hybridoma cells. using hybridoma technology, we obtained 3 hybridoma cell lines that could stably secrete A. acidoterrestris antibody. Two of the three were IgG1 and their biological characteristics were identified. [Results] Two monoclonal antibodies 3F7 and 9C4 had different antigenic sites and the stability remained unchanged after multiple passages. Specificity experiments showed that the two monoclonal antibodies did not cross react with A. acidocaldarius (NCIMB11725), Bacillus cereus (ATCC11778), Bacillus subtilis (ATCC11774) and A. cycloheptanicus (ATCC49029). [Conclusion] These two monoclonal antibodies can be further used in the development of colloidal gold test strips.