A highly sensitive and stable electrochemical sensor was developed by modifying a pencil graphite electrode (PGE) with nitrogen-doped carbon dots and gold nanoparticles (AuNPs@N-CDs). This hybrid-modified electrode exhibited significantly enhanced electrochemical performance for detecting ciprofloxacin (CIP) compared to the bare PGE and electrodes modified with either N-CDs or AuNPs alone. The morphology and structural properties of the AuNPs@N-CDs/PGE were comprehensively analyzed using scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX). Electrochemical methods, including cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were used to investigate the electrode modification process, assess CIP concentration, and examine potential interference effects. The sensor demonstrated a broad linear detection range from 5.0 × 10⁻10 to 3.0 × 10⁻⁸ M, with a detection limit reaching 4.3 × 10⁻11 M. Moreover, the sensor was effectively utilized for the quantitative analysis of CIP in real-world samples such as human serum, urine, and pharmaceutical tablets, confirming its suitability for trace-level detection in real sample.