Gamete release, an essential event for animals, is regulated by various factors including environmental stimuli, neural circuit, and endocrine molecules. In this study, we investigated the mechanisms regulating gamete release in the ascidian Ciona robusta, part of the sister group of vertebrates. Ciona is a hermaphrodite, releasing sperm first from orange-pigmented organ (OPO) at the end of the spermiduct, followed by eggs from the oviduct beneath the spermiduct knob-like structure. In this study, behavioral and morphological analyses revealed that this sequential release occurs because the knob-like structure compresses the oviduct opening at the onset of gamete release. Observations of transgenic Ciona expressing Kaede under the gonadotropin-releasing hormone 2 (Gnrh2) promoter showed that Gnrh2-expressing neurons and fibers accumulate in the epithelium around the spermiduct openings. In contrast, Gnrh1-expressing neurons are localized in the cerebral ganglion and project toward the ovary, suggesting distinct roles of GnRH1 and GnRH2 in reproductive regulation. RNA-seq and real-time PCR analyses revealed that Opsin2, Opsin3, beta-carotene-15,15\'-monooxygenase (Bco), and several ion channel genes are specifically expressed in the OPO, along with Gnrh2. In situ hybridization showed that these genes are localized in the innermost OPO layer, suggesting that the OPO functions as a photoreceptive organ. Although, Gnrh2 expression has been considered low in adult Ciona, our study revealed strong OPO-specific expression with photoreceptive genes. Collectively, these findings suggest that GnRH2 plays a central role in gamete release regulation, potentially in coordination with the photoreceptive system, while GnRH1 may regulate ovarian functions. These results indicate that C. robusta employs distinct GnRH systems in a tissue-specific manner to regulate reproduction.