It is imperative to discriminate between male and female fish during the juvenile stage and pre-adjust the required numbers of males and females to efficiently raise parent fish in aquaculture farms. One method to discriminate between males and females is to measure a fish-specific steroid hormone such as 11-ketotestosterone (11-KT) in fish plasma. However, conventional methods such as enzyme-linked immunosorbent assay (ELISA) are complicated to operate and require a long time for analysis. Here, we introduce a novel immunosensor system that combines immunological reaction and electrochemical measurement with the aim of achieving a rapid and straightforward measurement method in preparation for gender determination of fish. Using our system, we observed stepwise decreases in the oxidation peak current value with increasing concentration of 11-KT in the sample. Furthermore, a strong linear relationship was obtained between the standard solution concentrations of 0.21 and 3.30 pmol ml−1 (R = 0.9869) in only 20 min. Next, the optimum operating conditions of the sensor were evaluated. It was also confirmed that the sensor did not respond to other hormones. Moreover, a strong linear relationship was obtained between the values and those obtained by the conventional ELISA method (R = 0.9927).