Temporal changes in the plasma total cholesterol concentration of fish are an indication of fish health, and a continuous in vivo monitoring method for total cholesterol is therefore desirable. An enzyme biosensor would be a good choice for continuous measurement. However, a biosensor as a foreign substance has a low sensitivity after a certain period of measurement owing to immune system attack. We introduced a highly sensitive carbon nanotube (CNT) to enhance sensor sensitivity for the real-time monitoring of total cholesterol in free-swimming fish (Oreochromis niloticus). The sensor was constructed using a Pt-Ir electrode that was dipped into a dispersed CNT solution and dried to adsorb the CNT onto the electrode. Cholesterol esterase, cholesterol oxidase, and a mediator were then immobilized on the sensor. The proposed sensor output current correlated well (R = 0.9992) with a cholestreyl oleate standard (10–300 mg dl−1). The dynamic range matched the range of cholesterol concentrations in fish (50–300 mg dl−1). A sensor with a mediator was not affected by changes in oxygen concentration. Changes in total cholesterol concentration could be continuously monitored in free-swimming fish using this sensor for 26 h.