Background <p>Pigeonpea is the second-most significant legume crop grown in tropics and subtropics. A substantial global concern lowering the yields of important crops like pigeonpea is disease-related damage which is accounting for major crop loss. Pigeonpea infestation with numerous insect pests and diseases result in reduced productivity. Among these Alternaria blight disease caused by <i>Alternaria alternata</i> fungi is one among the factors leading to decreased productivity. At micro-molar concentrations, fungal and bacterial growth can be inhibited by plant defensins, which are short, basic peptides rich in cysteine.</p> Methods and results <p>In the present investigation, two defensins <i>Tfgd2</i> (<i>Trigonella foenum-graecum</i> defensin 2) and <i>RsAFP2</i> (<i>Raphanus sativus</i> antifungal protein 2) combined by a linker peptide was incorporated as a fusion gene into pigeonpea through <i>Agrobacterium</i> mediated transformation. T₀ transgenic plants were analyzed by PCR and the positive plants were determined across the subsequent generations depending on the fusion gene inclusion, expression and bioefficiency. PCR, RT-PCR and Southern assay confirmed the stable incorporation and fusion gene expression in T₁ and T₂ transgenic pigeonpea plants. In disease resistance assay compared to the untransformed control plants fusion gene expressing plants showed enhanced disease resistance against Alternaria leaf blight with reduced leaf lesion size, ranging from 3.2 to 8.4 cm², whereas in the control it is 24.0 cm².</p> Conclusion <p>Our findings illustrate the promising activity of <i>Tfgd2</i>-<i>RsAFP2</i> fusion gene in conferring Alternaria blight disease resistance in transgenic pigeonpea and the same can be employed in the further development of disease resistant transgenic lines in pigeonpea improvement studies.</p>