Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.