Plant defensins and defensin like protein (DEFL) form a large family of small cysteine-rich proteins. They are major components of plant immune systems, being involved in host defenses against biotic and abiotic stresses. In this study, a novel defensin like protein (DEFL) gene PnDEFL1 was isolated from Panax notoginseng, a traditional Chinese medicinal herb. The expression patterns of PnDEFL1 after treatment with methyl jasmonate, salicylic acid, ethephon, and H2O2, as well as during Fusarium solani infection, were analyzed using reverse transcription qPCR. The up-regulated expression of PnDEFL1 indicated that it responded to F. solani infection and all four defense-related signalling molecules. The PnDEFL1 gene was further fused with the green fluorescent protein gene in a plant expression vector and transformed into onion (Allium cepa) epidermal cells. The laser scanning confocal microscope confirmed that the PnDEFL1 protein localized to the extracellular region. In addition, the recombinant PnDEFL1 protein was expressed in Escherichia coli and purified by affinity chromatography. It had antifungal activities against F. solani, F. oxysporum, Botrosphaeria dothidea, and Sclerotinia sclerotiorum. The PnDEFL1 gene was transferred into tobacco (Nicotiana tabacum) to verify its function. The overexpression of PnDEFL1 in tobacco conferred a high resistance to F. solani infection. Thus, the PnDEFL1 gene is involved in the defense responses of P. notoginseng to F. solani infection.