Canine apocrine gland anal sac adenocarcinoma (AGASACA) is a rare, malignant tumor in dogs. To date, few cell lines are available and used to establish the current treatment protocols. Organoids are three-dimensional cell cultures derived mainly from stem cells and can reproduce tissue's epithelial structure, function, and genetics, and thus, of great promise in precision medicine. In the current investigation, 10 AGASACA organoid lines were developed from surgically removed tissues of AGASACA-affected dogs and analyzed for comparison with the original tissues. AGASACA organoids were successfully generated from all cases and were positive for CK7, HER2, p53, p63, VEGF, and Ki67, and negative for CK20, consistent with previous reports in dogs and humans. Electron microscopic imaging of AGASACA organoids showed organelles, including numerous granules and fat droplets that characterize apocrine gland cells. AGASACA organoids were tumorigenic in vivo in immunodeficient mice. In addition, treatment of the AGASACA organoids with carboplatin, mitoxantrone, toceranib, and lapatinib revealed different sensitivity profiles among lineages, with carboplatin and lapatinib, in particular, being divided into sensitive and resistant ones. In contrast, mitoxantrone and toceranib showed generally high efficacy in all organoids. In conclusion, our established AGASACA organoids have the potential to be an experimental tool for the development of novel therapies for canine and human apocrine gland adenocarcinoma.