Here, we developed extended-gate field-effect transistor (EG-FET)-based chemosensors with molecularly imprinted polymers (MIPs) on a gold-dendritic (AD) electrode to detect tumor necrosis factor-α (TNF-α) in serum. The poly(β-cyclodextrin) film was electrochemically prepared on the AD electrode using a potentiodynamic technique, followed by TNF-α immobilization by host-guest interaction. The MIP film was formed via electropolymerization of thiophene-3-amidoxime (T3A) monomer in PBS solution with one scan cycle at an applied voltage of 0–1.2 V. After electrochemical optimization, the sensing behavior (based on drain current, Ids) of the MIP films was investigated to explore the validity of the sensors, resulting in excellent reproducibility, reusability, and stability. Based on the ΔIds – CTNF-α regression curves obtained in serum containing various analyte concentrations, the imprinting factor (IF) of MIP-based EG-FET sensor was 5.55. The selectivity was evaluated by comparing sensing property using analogous cytokine proteins (interleukin 1β [IL-1β] and interleukin-6 [IL-6]). The MIP-based EG-FET sensors exhibited high sensitivity (LOD: 0.55 pg/mL, LOQ: 1.82 pg/mL) and excellent selectivity (coefficient (α)> 3). Based on the excellent sensing performances, including high sensitivity and selectivity, excellent reproducibility, robustness, reusability, and stability, our (EG-FET)-based chemosensor with TNF-α-recognizing MIP film can be used for the early diagnosis and point–of–care of immune-related diseases.