Despite the excellent diagnostic applications of the current conventional immunoassay methods such as ELISA, immunostaining and Western blot for FAM134B detection, they are laborious, expensive and required a long turnaround time. Here, we report an electrochemical approach for rapid, sensitive, and specific detection of FAM134B protein in biological (colon cancer cell extracts) and clinical (serum) samples. The approach utilises a differential pulse voltammetry (DPV) in the presence of the [Fe(CN)6]3−/4− redox system to quantify the FAM134B protein in a two-step strategy that involves (i) initial attachment of FAM134B antibody on the surface of extravidin-modified screen-printed carbon electrode, and (ii) subsequent detection of FAM134B protein present in the biological/clinical samples. The assay system was able to detect FAM134B protein at a concentration down to 10 pg μL−1 in phosphate buffered saline (pH 7.4) with a good inter-assay reproducibility (% RSD = <8.64, n = 3). We found excellent sensitivity and specificity for the analysis of FAM134B protein in a panel of colon cancer cell lines and serum samples. Finally, the assay was further validated with ELISA method. We believe that our assay could potentially lead a low-cost alternative to conventional immunological assays for target antigens analysis in point-of-care applications.