<p>This study investigated the effects of dietary soybean isoflavones (SI) on ovarian development, hemolymph hormone and vitellogenin levels, and ovarian mitochondria-related indices in female <i>Macrobrachium rosenbergii</i>. A total of 600 prawns with an initial body weight of approximately 13&#xa0;g were randomly allocated to five groups, with four replicates per group and 15 females and 15 males per replicate. The prawns were fed diets containing graded levels of 0.88, 2.68, 4.42, 8.84, and 14.98&#xa0;mg/kg SI (designated as SI0.88, SI2.68, SI4.42, SI8.84, and SI14.98, respectively) for 56&#xa0;days. Compared with the SI0.88 group, the SI8.84 group showed a significantly higher gonadosomatic index and elevated hemolymph estradiol (E₂), progesterone (PROG), and vitellogenin (VTG) levels (<i>P</i> &lt; 0.05). Histological observations showed that the oocyte diameter in the SI8.84 group was significantly greater than that in all other experimental groups (<i>P</i> &lt; 0.05). Ultrastructural observations further indicated that, compared with the SI0.88 group, ovarian mitochondria in the SI8.84 group had relatively intact double-membrane structures and well-preserved cristae. In addition, ovarian ATP content and cytochrome c oxidase activity were significantly higher in the SI8.84 group than in all other groups, whereas ovarian reactive oxygen species (ROS) levels were significantly lower in the SI8.84 and SI14.98 groups than in the remaining groups (<i>P</i> &lt; 0.001). Transcriptomic analysis of ovarian tissues from stage V of female <i>M. rosenbergii</i> between the SI8.84 and SI0.88 groups identified 226 differentially expressed genes, mainly enriched in the PPAR signaling pathway, cholesterol metabolism, and other metabolism-related pathways. Selected genes, including <i>MrLAG</i> and <i>SMYD3-like</i>, were significantly upregulated in the SI8.84 group (<i>P</i> &lt; 0.05). In conclusion, appropriate dietary SI supplementation was associated with improved ovarian developmental status, accompanied by favorable changes in hemolymph hormone and vitellogenin levels, ROS levels, and mitochondria-related indicators. Regression analyses on hemolymph and ovarian indicators estimated the suitable dietary SI supplementation level for female <i>M. rosenbergii</i> to be 8.42–11.17&#xa0;mg/kg under the present experimental conditions. Collectively, these findings demonstrated the use of SI as a functional feed additive to support ovarian development in female <i>M. rosenbergii</i>, supporting its use as a functional feed additive in broodstock nutritional management.</p>