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A graphitic carbon nitride functionalized gold nanoparticles (g-C 3 N 4 /AuNPs) based fluorometric assay was developed for simple and rapid on-site amikacin determination. g-C 3 N 4 fluorescence is reduced (turn-off) when they bind to AuNPs but recovers (turn-on) when amikacin binds to AuNPs and releases the g-C 3 N 4. The assay is based on the effective property of AuNPs as absorbers in inner filter effect (IFE) systems. Under optimized conditions of 25 mM Na 2 SO 4 , pH 6.0, 3.0 mg mL−1 g-C 3 N 4, and incubation time of 12 min, the proposed method achieved a low detection limit of 5 × 10−9 M (2.9 ppb), wide linear range from 10−8 to 10−6 M, high precision (RSD <5 %) and high selectivity over other ions including Ca2+, Mg2+, Fe2+, NO 3 −, and PO 4 3− as well as several antibiotics, namely naproxen, levofloxacin, vancomycin, ceftriaxone, amoxicillin. The fluorometric sensor based on g-C 3 N 4 /AuNPs has produced good agreement quantifying amikacin in pharmaceutical samples, as cross-checked with LC-MS/MS method. The developed fluorometric sensor is promising for rapid on-site detection of other organic and inorganic substances with high sensitivity, selectivity, and minimal reagent consumption. • g-C 3 N 4 /AuNPs were synthesized and confirmed by XRD, IR, UV–Vis, TEM and SEM/EDS. • The recovery in fluorescence intensity of g-C 3 N 4 /AuNPs witnessed a linear increase with amikacin concentration. • A large linear range from 10−8 to 10−6 M, high accuracy, selectivity and low LOD of 5 × 10−9 M (2.9 ppb) of amikacin. • The proposed method was successfully applied for amikacin determination in pharmaceutical samples. [ABSTRACT FROM AUTHOR]