Two-dimensional materials are promising candidates for electrode modified materials in electrochemical sensing applications. This study introduces a non-enzymatic electrochemical sensor that combines tungsten disulfide (WS2) with fluorine-doped graphitic carbon nitride (F-g-C3N4) for the detection of acetaminophen (ACMP). The synthesized WS2/F-g-C3N4 microcomposite combines the two-dimensional metal dichalcogenide and the two-dimensional fluorine doped polymeric semiconductor to form the architecture of WS2 microspheres anchored on stacked F-g-C3N4 microflakes. Compared with the previously reported electrode modification materials for electrochemical detection of ACMP, the WS2/F-g-C3N4 microcomposite exhibits enhanced current response and high intrinsic sensitivity without employing any signal amplification strategy. This enhanced performance can be attributed to the synergistic combination of high conductivity, abundant catalytic sites, exceptional chemical stability, and significant effective surface area. Under optimal conditions, the WS2/F-g-C3N4/GCE displays a good analytical performance, including good linearity across two concentration ranges (0.01–253.3 & 253.3–1616.2 µM), an extremely low limit of detection (0.004 µM), and a good sensitivity (0.535 µA µM−1 cm−2). Additionally, it demonstrates good selectivity, reproducibility, repeatability, stability, and reusability for ACMP detection in the presence of various potential interferents. The WS2/F-g-C3N4/GCE is successfully applied to detect ACMP in pharmaceutical and environmental samples, achieving the recovery rates of 95.00–98.20%. This research provides an efficient and convenient approach for synthesis of 2D materials, which show great potential in electrochemical analysis.