To avoid fish mortality and improve productivity, the physiological conditions including stress state of the cultured fish must be monitored. As an important indicator of stress, glucose concentrations are monitored using in vitro blood analysis. The physiological processes of fish under environmental conditions are harsher in many ways than those experienced by terrestrial animals. Moreover, the process of anaesthetizing and capturing the fish prior to analysis may produce inaccurate results. To solve these problems, we developed wireless biosensor system to monitor the physiological condition of fish. This system enables artificial stress-free and non-lethal analysis, and allows for reliable real-time monitoring of fish stress. The biosensor comprised Pt–Ir wire as the working electrode and Ag/AgCl paste as the reference electrode. Glucose oxidase was immobilized on the working electrode using glutaraldehyde. We used the eyeball interstitial sclera fluid (EISF) as the in vivo implantation site of the sensor, which component concentration correlates well with that of blood component concentration. In the present study, we investigated stress due to alterations in water chemistry, including dissolved oxygen, pH, and ammonia–nitrogen compounds. Stress perceived from behavioural interactions, including attacking behaviour and visual irritation, was also monitored. Water chemistry alterations induced increases in the glucose concentration (stress) that decreased with removal of the stimulus. For behavioural interactions, stress levels change with avoidance, sensory behaviour and activity. We believe that the proposed biosensor system could be useful for rapid, reliable, and convenient analysis of the fish physiological condition and accurately reflects the stress experienced by fish.