In this study, we focused on nitric oxide (NO), which is now being elucidated as the dominant factor in nitrous oxide (N₂O) production by ammonia-oxidizing bacteria (AOB). In order to contribute to the development of technology for reducing N₂O production in full-scale wastewater treatment plants, we experimentally verified the possibility of extending the scope of the basic theory of N₂O production control by utilizing NO as the engineering indicator to complex microbial systems including denitrifying bacteria as well as AOBs. We designed the simple method for testing NO and N₂O production using disposable syringes and tested activated sludge collected from a wastewater treatment plant. The results showed that NO production is the dominant factor in the conversion rate and production rate of N₂O indicated by activated sludge in anaerobic, aerobic, and anoxic all biological reaction tanks. These results indicate the possibility of controlling N₂O production by comprehensively observing the NO-mediated interactions of complex microbial systems, such as nitrifying and denitrifying bacteria.