Intracellular polysulfide could regulate the redox balance via its anti-oxidant activity. However, the existence of polysulfide in biological fluids still remains unknown. Recently, we developed a quantitative analytical method for polysulfide and discovered that polysulfide exists in plasma and responds to oxidative stress. In this study, we confirmed the presence of polysulfide in other biological fluids, such as semen and nasal discharge. The levels of polysulfide in these biological fluids from healthy volunteers (n = 9) with identical characteristics were compared. Additionally, the circadian rhythm of plasma polysulfide was also investigated. The polysulfide levels detected from nasal discharge and seminal fluid were approximately 400 and 600 μM, respectively. No correlation could be found between plasma polysulfide and the polysulfide levels of tear, saliva, and nasal discharge. On the other hand, seminal polysulfide was positively correlated with plasma polysulfide, and almost all polysulfide contained in semen was found in seminal fluid. Intriguingly, saliva and seminal polysulfide strongly correlated with salivary amylase and sperm activities, respectively. These results provide a foundation for scientific breakthroughs in various research areas like infertility and the digestive system process.