Here, we report a chromosome-level genome assembly of Talaromyces rugulosus (syn. Penicillium rugulosum) W13939 (six chromosomes; contig N50: 5.90 Mb), generated using a combination of PacBio long-read and Illumina paired-end data. T. rugulosus is not only a potent enzyme producer, but also a mycoparasite of Aspergillus flavus, which is a notorious plant pathogen and mycotoxin producer, making it a promising biocontrol agent. The T. rugulosus genome is rich in genes encoding proteases, carbohydrate-active enzymes, fungal cell wall-degrading enzymes, lectin, and secondary metabolite biosynthetic enzymes, reflecting its mycoparasitic life style and mycotoxigenic capability. This high-quality assembly of the T. rugulosus genome will be a valuable resource to assist us in the understanding of the molecular basis of mycoparasitism and facilitate the agricultural and biotechnological applications of Talaromyces spp.