Fish display a wide variation in their physiological responses to stress, which is clearly evident in plasma corticosteroid changes, chiefly the cortisol level, in fish. Cortisol is a well-known indicator of fish stress; thus, a simple and speedy approach to detecting plasma cortisol should be developed. Here we described a novel disposable carbon-nanotube-enhanced label-free immunosensor for detecting plasma cortisol level. The principle of the sensor system is based on differences in the electrochemical change in oxidation peak current induced by an immunoreaction that depends on the cortisol level of the sample. We used a single-walled carbon nanotube to improve the sensitivity and conductivity of the system. The proposed sensor system showed a linear correlation (R = 0.992) between cortisol level and oxidation peak current in the range from 156 to 10000 pg ml−1. The specificity of the label-free immunosensor system was investigated using other steroid hormones; the specificity of cortisol was suggested by a minimal change in the oxidation peak current of the other steroid hormones. The sensor system was used to determine the plasma cortisol level of Nile tilapia (Oreochromis niloticus) and the results were compared with those of the same samples determined using a conventional method (ELISA). A very good correlation was obtained between results determined using both methods (correlation coefficient, 0.999).