Pharmaceutical effluents are a serious environmental issue, which require to be treated by a suitable technique; thus, the electrochemical process is actively considered as a viable method for the treatment. In this work, new carbon paste electrodes (CPEs) were fabricated by compressing gold and silver nanoparticles (NPs), namely, CPE/Ag NPs, CPE/Au NPs, and CPE/Ag/Au NPs and then completely characterized by different analytical methods. The performance of the electrodes was studied after determining their surface area (×10-6 cm2) as 4.17, 5.05, 5.27, and 5.12, producing high anodic currents for K4[Fe(CN)6] compared to the commercial electrode. This agrees with the results of impedance study, where the electron transfer rate constants (kapp, ×10-3 cm s-1) were determined to be 28.7, 42.6, 41.0, and 101.4 for CPE, CPE/Ag NPs, CPE/Au NPs, and CPE/Ag/Au NPs, respectively, through the Bode plot-phase shifts. This is consistent with the charge transfer resistance (RCT, Ω), resulting as 171 for CPE/Ag/Au NPs < 395 for CPE/Ag NPs < 427 for CPE/Au NPs and < 742 for CPE. Therefore, these electrodes were employed to detect trimethoprim (TMP) since metallic NPs contribute good crystallinity, stability, conduciveness, and surface plasmon resonance to the CPE, convalescing the sensitivity; comprehensively, they were applied for its detection in real water and human urine samples, and the limit of detection (LOD) was as low as 0.026, 0.032, and 0.026 μmol L-1 for CPE/Ag NPs, CPE/Au NPs, and CPE/Ag/Au NPs, respectively. In contrast, unmodified CPE was unable to detect TMP due to the lack of efficiency. The developed technique shows excellent electrochemical recovery of 92.3 and 97.1% in the urine sample. Density functional theory (DFT) was used to explain the impact of the metallic center in graphite through density of states (DOS).