Like humans, cancer affects companion animals with similar genetic risks and incident rates. To improve treatment strategies for pet cancers, new research models are necessary. Patient-derived 3D organoid culture models are valuable and ensure the development of new effective therapies. In the previous study, we established a 3D organoid-derived 2.5D organoid culture model that recapitulated some characteristics of their parental 3D organoids. In the present study, we aimed to generate a 2.5D organoid culture model directly from cancer-diseased dogs and cats using special 2.5D media. The primary cultured cells in 2.5D media (direct 2.5D organoids) showed better attachment, growth, marker expression, and faster proliferation speed than those cultured in normal Dulbecco’s Modified Eagle Medium media. The direct 2.5D organoids showed expression of each specific marker to their original cancer tissues and exhibited tumorigenesis in vivo. Moreover, the direct 2.5D organoids exhibited concentration-dependent responses to anti-cancer drugs, and different sensitivity profiles were shown among the strains. Our data suggest that the direct 2.5D organoid culture model might become a useful tool beyond 2D cell lines to study cancer biology in companion animals and could provide new platforms for screening the anti-cancer drugs.