This paper reports the development of a novel electrochemical platform based on graphite electrodes modified with copolymer poly(aniline-4-aminophenol) and anti-triiodothyronine, integrated into the design of an electrochemical immunosensor for thyroid hormone, allowing free triiodothyronine (T3) detection in real serum samples. The proposed copolymer showed synergistic characteristics among its original polymers, which led to good results in the biofunctionalization step. A specific antibody for T3 was immobilized on copolymer poly(aniline-4-aminophenol). In the presence of target T3, an immunological reaction occurs with anti-T3, being observed a current decrease proportional to T3 concentration. The decrease was monitored by differential pulse voltammetry, detecting up to 0.60 pg mL −1 and showing good stability, maintaining 90% of its response after 60 days of storage. This platform shows linear relationship between the anodic current and T3 concentration with linear regression as y = 616.79 − 26.03 x ( r 2 = 0.9976). The immunosensor shows suitable for detection of thyroid hormones in real serum samples, as well as potential for use in detection of other disease biomarkers.