Stress can force the farmed animals into a pathological condition and disease. Tryptophan derived monoamine neurotransmitters play an essential role in the regulation of neural activity. To understand the role of tryptophan metabolism in flatfish stress response, we investigated the functional roles of tryptophan and its main metabolites input on the Dahlgren cell population in the caudal neurosecretory system (CNSS) of olive flounder. Ex vivo electrophysiological recordings demonstrated that both tryptophan and all tested metabolites increased the electrical activity of Dahlgren cells, which was supported by elevated mRNA levels of ion channels and major secretion hormone genes in CNSS. Both tryptophan and the tested metabolites function as excitatory neuromodulators on Dahlgren cells, with a sensitivity sequence to Dahlgren cells observed as follows: serotonin > melatonin > tryptophan > kynurenine. Additionally, the mRNA expression of key genes in serotonin - melatonin metabolic pathway significantly increased after tryptophan and serotonin treatment. Collectively, our results provide direct evidence that both tryptophan and its main metabolites can act as an excitatory neuromodulator on Dahlgren cells and mediate stress regulation primarily through the tryptophan - serotonin - melatonin metabolic pathway in flatfish.