Antheraea pernyi Guérin-Méneville is used for silk production and as a food resource. Its infection by exogenous pathogens, including microsporidia, fungi, bacteria, and virus, can lead to silkworm diseases, causing major economic losses. A trypsin-like serine protease gene (TLS) was found in A. pernyi transcriptome data resulting from two different infection experiments. The cDNA sequence of ApTLS was 1,020 bp in length and contained an open reading frame of 774 bp encoding a 257-amino acid protein (GenBank KF779933). The present study investigated the expression patterns of ApTLS after exposure to different pathogens, and in four different A. pernyi strains. Semiquantitative RT-PCR indicated that ApTLS was expressed in all developmental stages and was most expressed in the midgut. Quantitative real-time PCR indicated ApTLS was upregulated in the midgut of A. pernyi exposed to nucleopolyhedrovirus (ApNPV), Nosema pernyi, Enterococcus pernyi, and Beauveria bassiana infections, and the highest gene expression level was found under ApNPV infection. The strain Shenhuang No. 2 presented the lowest infection rate and the highest ApTLS gene expression level when exposed to ApNPV. Thus, ApTLS seems to be involved in innate defense reactions in A. pernyi.