In this study, graphene quantum dots (GQDs) were employed for quantitatively analyzing lamivudine using a fluorescence quenching technique. This approach allows for sensitive determination of the concentration of lamivudine in different matrices without requiring derivatization. The mechanism behind the fluorescence intensity quenching between GQDs and lamivudine molecules was explored using the Stern Volmer equation, revealing dynamic quenching behavior. Additionally, various factors affecting fluorescence quenching efficiency such as pH, GQDs concentration, and incubation time were carefully tuned. Moreover, our developed method successfully met ICH guidelines for validation parameters including linearity, accuracy, precision, and selectivity demonstrating excellent performance. The results showed good accuracy and precision, with a mean recovery value of 101.91% for method accuracy and a relative standard deviation of 0.682 and 1.489 for intraday and interday precision, respectively. Finally, the greenness and blueness of the developed method were also investigated to assess its environmental friendliness and analytical practicality. Greenness evaluation using the AGREE tool demonstrated that the developed method has a low environmental impact with an AGREE score of 0.75, Besides, the blueness evaluating using the BAGI tool indicated that the developed method is practical, reliable, and well-suited for routine analysis of lamivudine in various samples.