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The increase in anthropogenic pollution raises serious concerns regarding contamination of water bodies and aquatic species with potential implications on human health. Pharmaceutical compounds are a type of contaminants of emerging concern that are increasingly consumed and, thus, being frequently found in the aquatic environment. In this sense, an electrochemical sensor based on an unmodified and untreated carbon fiber paper (CPS—carbon paper sensor) was simply employed for the analysis of trimethoprim antibiotic in fish samples. First, the analytical conditions were thoroughly optimized in order for the CPS to achieve maximum performance in trimethoprim determination. Therefore, an electrolyte (0.1 M Britton–Robinson buffer) pH of 7 was selected and for square wave voltammetry parameters, optimum values of amplitude, frequency and step potential corresponded to 0.02 V, 50 Hz, and 0.015 V, respectively, whereas the deposition of analyte occurred at +0.7 V for 60 s. In these optimum conditions, the obtained liner range (0.05 to 2 µM), sensitivity (48.8 µA µM−1 cm−2), and LOD (0.065 µM) competes favorably with the commonly used GCE-based sensors or BDD electrodes that employ nanostructuration or are more expensive. The CPS was then applied for trimethoprim determination in fish samples after employing a solid phase extraction procedure based on QuEChERS salts, resulting in recoveries of 105.9 ± 1.8% by the standard addition method. [ABSTRACT FROM AUTHOR]