Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.