The rate and extent of drug release under physiological conditions is a key factor influencing the therapeutic activity of a formulation. Real-time detection of drug release by conventional pharmacokinetics approaches is confounded by low sensitivity, particularly in the case of tissue-targeted novel drug delivery systems, where low concentrations of the drug reach systemic circulation. We present a novel fluorescence turn-on platform for real-time monitoring of drug release from nanoparticles based on reversible fluorescence quenching in fluorescein esters. Fluorescein-conjugated carbon nanotubes (CNTs) were esterified with methotrexate in solution and solid phase, followed by supramolecular functionalization with a chemoenhancer (suramin) or/and a stealth agent (dextran sulfate). Suramin was found to increase the cytotoxicity of methotrexate in A549 cells. On the other hand, dextran sulfate exhibited no effect on cytotoxicity or cellular uptake of CNTs by A549 cells, while a decrease in cellular uptake of CNTs and cytotoxicity of methotrexate was observed in macrophages (RAW 264.7 cells). Similar results were also obtained when CNTs were replaced with graphene. Docking studies revealed that the conjugates are not internalized by folate receptors/transporters. Further, docking and molecular dynamics studies revealed the conjugates do not exhibit affinity toward the methotrexate target, dihydrofolate reductase. Molecular dynamics studies also revealed that distinct features of dextran-CNT and suramin-CNT interactions, characterized by π–π interactions between CNTs and dextran/suramin. Our study provides a simple, cost-effective, and scalable method for the synthesis of nanoparticles conferred with the ability to monitor drug release in real-time. This method could also be extended to other drugs and other types of nanoparticles.