The determination of the concentration of acetaminophen (AC) in the human body is important due to the regular drug safety and public health. However, conventional methods are insufficient because of their expensive, time-consuming and complicated nature. In this study, a novel two-step activation of biomass-derived carbon for the electrochemical detection of AC is presented. The electrode material is prepared by a novel two-step activation process, which involves the initial activation of kelp powder with ZnCl₂, subsequently by an activation step with KOH. The activation procedure greatly enhanced the overall pore volume and specific surface area. The characterizations of ZnCl₂-KOH activated kelp carbon (ZKAKC) were conducted with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, Brunauer-Emmett-Teller (BET) surface area analysis and electrochemical impedance spectroscopy (EIS). The electrochemical characterization of ZKAKC was performed with cyclic voltammetry (CV) analysis of potassium ferricyanide. The detection ability of ZKAKC/GCE toward AC was conducted using CV analysis and differential pulse voltammetry (DPV). The modified electrode showed high sensitivity and selectivity for the determination AC with the detection limit of 0.004 μM. Also, the modified electrode showed good result toward AC even in the presence of 0.1 mM ascorbic acid and 0.1mM dopamine with the detection limit of 0.007 μM. For the evaluation of sensing ability as an actual electrochemical sensor, a real sample test was conducted. The high electrochemical performance was due to the increased physical and electrochemical surface area, which occurred during the two-step activation process. This approach for producing activated carbon is universal for development of new carbon materials and can be applied to different carbon source materials.