Polychlorinated biphenyls (PCBs) are a highly toxic family of synthetic chemical compounds. PCBs are widely spread in the environment and their toxicity can cause serious ailments to living organisms such as cancer; therefore, developing a device for the detection of PCBs in the environment is significant. In this paper, polyclonal primary anti-PCB antibodies were immobilized onto a gold screen-printed electrode with the purpose of creating an electrochemical immunosensor for the detection of Aroclor 1254. It was modified with 11-mercaptoundecanoic acid (11-MUA) and the activation of the carboxylic acid terminal was performed by cross-linking 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hyrodsuccinmide (NHS) on the electrode surface. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), linear sweep voltammetry, atomic force microscopy (AFM), scanning electron microscopy (SEM), and contact angle measurement were employed to characterize SAM development on the gold electrode. Using a competitive assay, a 0.09 ng/mL−1 limit of detection and a linear range of 0.101–220 ng/mL−1 were determined. The self-assembled monolayers (SAM) were successful in encapsulating the PCBs on the immunosensor. The electrochemical detection showed better resolution when compared to traditional methods such as the ELISA optical technique. The novel electrochemical immunosensor approach that is discussed in this paper has the potential to offer rapid sample screening in a portable, disposable format and could contribute to the effective control and prevention of PCBs in the environment.