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Thaumatin-like proteins (TLPs) are involved in the plant defense response against pathogens, and most of them exhibit antifungal activity. However, the role of TLPs in pathogen-induced defense responses in spruce is not fully understood. In this study, four <i>TLP</i> genes encoding thaumatin-like protein, designated as <i>PlTLP1–4</i>, were isolated and identified from <i>Picea likiangensis</i> needles. Sequence analysis showed that <i>PlTLP1</i>, <i>PlTLP3</i>, and <i>PlTLP4</i> contained 16 conserved cysteine residues, while <i>PlTLP2</i> had only 10 conserved cysteine residues. qPCR analysis showed that <i>PlTLPs</i> were expressed in all tissues tested, <i>PlTLP1</i>, <i>PlTLP3</i>, and <i>PlTLP4</i> had the highest expression levels in young fruits, while <i>PlTLP2</i> had the highest expression levels in roots. In addition, the expression levels of four <i>PlTLPs</i> were significantly upregulated during infection by <i>Lophodermium piceae</i>. Four recombinant <i>PlTLPs</i> expressed in <i>Escherichia coli</i> exhibited obvious β-1,3-glucanase activity. The antifungal activity assay showed that four recombinant <i>PlTLPs</i> had significant inhibitory effects on the mycelial growth of <i>L. piceae</i>, <i>Fusarium proliferatum</i>, <i>Botrytis cinerea</i>, and <i>Roussoella doimaesalongensis</i>. Microscopic observation revealed that the recombinant <i>PlTLP1–4</i> induced the morphological changes of the mycelia of <i>L. piceae</i>, and the recombinant <i>PlTLP2</i> and <i>PlTLP3</i> induced the morphological changes of the mycelia of <i>F. proliferatum</i> and <i>R. doimaesalongensis</i>, while all the recombinant <i>PlTLPs</i> had no obvious negative effect on the morphology of <i>B. cinerea</i> mycelium. These results suggest that <i>PlTLP</i> genes may play an important role in the defense response of <i>P. likiangensis</i> against <i>L. piceae</i> invasion.