In this study, we report the development of a sensitive electrochemical immunosensor for the detection of the tumor necrosis factor-α (TNF-α) cytokine in human saliva samples. The fabricated immunosensor was based on gold substrates. The anti-TNF-α antibody (Ab-TNF-α) was electro-addressed onto the gold working electrodes (WEs) through functionalization with 4-carboxymethylaniline (CMA). Cyclic voltammetry (CV) was applied during the functionalization process to characterize the gold WEs surface properties. Finally, the chronoamperometry technique was applied to characterize the modified gold WEs. The use of a secondary antibody anti-TNF-α (Ab-TNF-α-HRP) labelled with horseradish peroxidase (HRP) was investigated using tetramethylbenzidine (TMB) as electrochemical substrate. A sandwich-type detection strategy was then employed for TNF-α detection through the labelled antibody Ab-TNF-α-HRP activity in a TMB solution. Under the optimal experimental conditions, the specificity of the immunosensor was investigated by analysing solutions containing possible salivary interferences represented by other molecules secreted in acute stage of inflammation such as interleukin-10 (IL-10) and the hormone cortisol. The developed immunosensor showed good performances for Ag-TNF-α cytokine detection within the range of 1 pg mL⁻¹–30 pg mL⁻¹ with a limit of detection (LOD) of 1 pg mL⁻¹. As there is a correlation between TNF-α and the severity of heart failure (HF), this new immunosensor may represent a promising bioanalytical tool for the HF monitoring by saliva analysis. Moreover, the CA technique provides analyses in 5 s for each concentration, which save time and provides rapid data to doctors and clinicians.