Display omitted •Activated carbon was prepared with a novel two-step activation process.•Kelp is used as source material for the biomass-derived activated carbon.•The modified electrode was used for the electrochemical determination of acetaminophen.•High sensitivity toward acetaminophen and a low detection limit were achieved. The determination of the concentration of acetaminophen (AC) in the human body is of significant importance to carefully monitor and regular drug safety and public health. However, most conventional methods are insufficient due to their time-consuming, expensive and complicated nature. In this study, we report a novel two-step activation of biomass-derived carbon for the electrochemical determination of AC. The electrode material is prepared by a two-step activation process, which involves the initial activation of kelp powder with ZnCl2, followed by an activation step with KOH. The activation procedure greatly increased 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 sensing ability of ZKAKC/GCE toward acetaminophen was conducted using CV analysis and differential pulse voltammetry (DPV). The modified electrode showed high sensitivity, selectivity and a good detection limit for the determination acetaminophen with the detection limit of 0.004 μM. Also, the modified electrode showed good result toward acetaminophen even in the presence of ascorbic acid and 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 electrochemical performance was enhanced due to the increased physical and electrochemical surface area, which occurred during the two-step activation process. This approach for producing activated carbon is crucial for future development and can be applied to different carbon source materials.