Gentamicin sulfate (GEN), a well-known broad-spectrum antibiotic is mostly administered through intramuscular injections and entirely excreted in un-metabolized form through urination from patient's body. Quantitative detection of GEN by direct UV absorption is usually challenging due to lack of chromophores and fluorophores in structure. The current study described the hesperidin coated silver nanoparticles (HSPAgNPs) based novel colorimetric quantitative assay for GEN. HSPAgNPs, based colorimetric detection involved a transition from characteristic yellow colour to blackish brown upon addition of GEN, accompanied by a significant quenching in localized surface plasmon resonance (LSPR) band at λmax 398 nm. Moreover, the synthesized HSPAgNPs were employed to rapid and quantitative detection of GEN in concentration range of 5 to 100 μM. Limit of detection (LOD) and limit of quantification (LOQ) was calculated by standard deviation of the ordinate intercept and slope of the regression line and estimated to be 6.89 μM and 20.88 μM respectively, with a linear correlation factor R2 equal to 0.9990 which strictly followed Beer's law. Furthermore, the utility and effectiveness of HSPAgNPs was also explored for selective recognition of GEN in tap water, serum, human blood plasma and urine.