The venom apparatus is a conserved organ in parasitoid wasps that shows adaptations correlated with life-style diversification. Recent venom analyses from selected species reveal considerable complexity and high diversity in venom composition existing not only between closely related species but even between strains and individuals, which may partly determine the potential for parasitoid adaptation. However, the investigations have paid little attention to secondary venom components that also have significant functions in parasitism, and the data in regard to full and accurate quantity of venom compositions at the protein level is not available. Using a combination of transcriptomic and label-free quantitative proteomic, we here explored the venom components of the endoparasitoid Tetrastichus brontispae (Eulophidae), a species devoid of polydnavirus, and provided an in-depth comparison of the venom proteomes between its two closely related strains, Tb-On and Tb-Bl. Results showed that approximately 1505 venom proteins were identified in the venom apparatus of T. brontispae, consistent with the classical venom protein characteristics, including enzymes, protease inhibitors, binding proteins and some immune related proteins. The venom extracts also contained novel venom proteins, such as kynurenine-oxoglutarate transaminase, 4-coumarate CoA ligase and venom protein r-like protein. Comparative venom proteomes revealed that significant quantitative and qualitative changes in venom composition occurred when Tb-Bl strain, with an invasive beetle Brontispae longissima pupa as its habitual host, was exposed to another invasive beetle Octodonta nipae pupa as host consecutively for two years; although the most abundant venom proteins were shared between them. These significantly differentially expressed proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response by enrichment analyses. Furthermore, most of the significantly enriched proteins presented strikingly increased levels or were exclusively identified in the Tb-On strain. These combined results indicated that virulence factors in Tb-On strain might be linked to lipid metabolism or more venom is required to inhibit O. nipae pupa’s melanotic encapsulation upon its parasitism. Altogether, our data reveal that venom composition can quickly evolve and respond to host selection, mainly through rapid changes in regulation of protein abundance and/or the emergence of multigenic families by gene duplication. Our data additionally provide invaluable data for further functional analysis of parasitoid venoms.