Reproduction is the basis of insect population growth and evolution, and encompasses ovarian development, reproductive behavior, and fecundity. Bactrocera dorsalis is a globally significant agricultural pest that is subject to quarantine, with mated females that can lay over 3000 eggs. The post-transcriptional regulation of ovarian development remains unclear. Here, miR-31b is shown to be involved in regulating Bactrocera dorsalis ovarian development. CRISPR/Cas9 was used to generate miR-31b loss-of-function mutations in Bactrocera dorsalis. The removal of miR-31b resulted in severely impaired ovarian development in adults, with phenotypes that included dramatically reduced egg production and hatching rates. The relationship between miR-31b and its target gene arylsulfatase B (ARSB) was subsequently identified using the methods of bioinformatics, transcriptomic sequencing, quantitative polymerase chain reaction (qPCR), RNA pull-down and dual-luciferase reporter assay. Finally, miR-31b was confirmed to bind the target gene arylsulfatase B to affect metabolism and thereby further hindered ovarian development of Bactrocera dorsalis. Overall, these results provide new insights into molecular mechanisms at the post-transcriptional level in regulating ovarian development and insect reproduction, consequently providing potential targets to control arthropod pests through the reproductive strategy. © 2024 Society of Chemical Industry.