Tobacco black shank (TBS), caused byPhytophthora nicotianae, is one of the most harmful diseases of tobacco. There are many studies have examined the mechanism underlying the induction of disease resistance by arbuscular mycorrhizal fungi (AMF) and β-aminobutyric acid (BABA) alone, but the synergistic effects of AMF and BABA on disease resistance have not yet been studied. This study examined the synergistic effects of BABA application and AMF inoculation on the immune response to TBS in tobacco. The results showed that spraying BABA on leaves could increase the colonization rate of AMF, the disease index of tobacco infected byP.nicotianaetreated with AMF and BABA was lower than that ofP.nicotianaealone. The control effect of AMF and BABA on tobacco infected byP.nicotianaewas higher than that of AMF or BABA andP.nicotianaealone. Joint application of AMF and BABA significantly increased the content of N, P, and K in the leaves and roots, in the joint AMF and BABA treatment than in the soleP. nicotianaetreatment. The dry weight of plants treated with AMF and BABA was 22.3% higher than that treated withP.nicotianaealone. In comparison toP. nicotianaealone, the combination treatment with AMF and BABA had increased Pn, Gs, Tr, and root activity, whileP. nicotianaealone had reduced Ci, H2O2content, and MDA levels. SOD, POD, CAT, APX, andPhactivity and expression levels were increased under the combined treatment of AMF and BABA than inP.nicotianaealone. In comparison to the treatment ofP.nicotianaealone, the combined use of AMF and BABA increased the accumulation of GSH, proline, total phenols, and flavonoids. Therefore, the joint application of AMF and BABA can enhance the TBS resistance of tobacco plants to a greater degree than the application of either AMF or BABA alone. In summary, the application of defense-related amino acids, combined with inoculation with AMF, significantly promoted immune responses in tobacco. Our findings provide new insights that will aid the development and use of green disease control agents.