Spiders are the most abundant venomous predators in the world. Previous research related to spider venom has mostly relied on transcriptomes and proteomes, with only a few high-quality genomes available. This is far from consistent with the species diversity of spiders. In this study, we constructed a high-quality chromosome-level genome assembly of Hylyphantes graminicola, which contained 13 chromosomes, with a genome length of 931.68 Mb and scaffold N50 of 77.07 Mb. Integrating genome, transcriptome, and proteome profiling, we identified a total of 59 coding genes among nine toxin gene families. Among them, Group 7 allergen (ALL7) protein was reported in spider venom for the first time. Its coding genes had a predicted signal peptide and maintained high expression levels in the venom, suggesting that ALL7 plays an important role in venom and maybe is a type of newly discovered venom toxin in the spider. By implementing comparative genomics, we found a similar gene number of main toxin gene families in spiders and the scorpion genome with conservative evolutionary rates, indicating that these toxin genes could be an ancient (~400 million years) and a conserved "basic toolkit" for spiders and scorpions to perform primary defence functions. Obtaining high-quality chromosome-level genomes from spiders not only facilitates venom research and toxin resource application, but also can improve comparative genomic analysis in other important traits, like the evolution of silk or behaviour.