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An immunosensor for the sensitive detection and estimation of tumor necrosis factor-alpha (TNF-α) in undiluted serum has been developed via an electrochemical enzyme-linked immunosorbent assay (ELISA) process. Electrochemical sensing was performed using a TNF-α specific monoclonal antibody modified self-assembled monolayer of dithiobis(succinimidyl propionate) on a comb-shaped gold electrode microarray. After anti-TNF-α antibody binding, unreacted active groups of DTSP were blocked using ethanol amine (EA) and nonspecific binding was prevented using phosphate buffer based starting block T20 (SB). Sensitive and disposable SB–EA–anti-TNF-α/DTSP/Au electrodes were exposed to solutions with different TNF-α concentrations for 20 min in undiluted serum. Conversion of 4-aminophenyl phosphate to 4-aminophenol and its electrochemical oxidation was utilized for indirect estimation of TNF-α. Results for SB–anti-TNF-α/DTSP/Au electrodes indicate that the sensors can be used for the sensitive estimation of TNF-α in undiluted serum in the range 500 pg/ml to 100 ng/ml with a detection limit of 60 pg/ml and sensitivity of 0.46 (ng/ml)−1. Negligible interference from serum and other biomarker proteins was observed. The described electrochemical ELISA is much faster than conventional ELISA and can be applied for sensing of a range of analytes in real patient samples.