In this paper, a novel poly-l-lysine (PLL) surface functionalized graphene quantum dots (GQDs) based sensor was developed for detection of cysteine (cys) and homo cysteine (hcys). A fluorescent probe (PLL-GQDs) was then fabricated by surface functionalizing GQDs with PLL, a biodegradable polycationic electrolyte to improve the sensitivity and selectivity towards cys and hcys. The detection was based on the specific binding of cys and hcys to PLL at the PLL-GQDs surfaces, which enabled dynamic quenching via electrostatic and hydrophobic interactions. This fluorescent probe provided good linearity for the tested biothiols, ranging from 0 to 150 nM for cys, from 0 to 100 nM for hcys, with limit of detections (LODs) of 2.38 and 1.94 nM, respectively in BPS (pH 7.4). Interestingly, fabricated probe was also able to display a significant selectivity towards cys and hcys against known interfering molecules. The cytotoxicity study confirmed the biocompatibility of PLL-GQDs, enabling its future scope for cell adhesion and other biomedical applications. Besides, confocal study revealed the excellent penetrations of PLL-GQDs into cell cytoplasm and nucleus that validate the practical application of developed probe to detect cys and hcys at cellular level. The method was successfully applied for detection of cys and hcys in human serum sample. We expect the design concept presented here would be broadly used for selective and sensitive estimation of cys and hcys. Display omitted •Waste material (pistachio shells) used for synthesis of graphene quantum dots (GQDs).•Novel poly-l-lysine (PLL) surface functionalized GQDs (PLL-GQDs) based sensor was developed.•The fabricated probe (PLL-GQDs) exhibited low cytotoxicity and excellent biocompatibility.•The probe demonstrated highly sensitive and selective detection of cysteine (cys) and homocysteine (hcys) in real samples.