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This manuscript presents a robust strategy for selective Pb(II) sensing based on a fluorescence turn‐off mechanism using ZnS quantum dots (QDs) biosynthesized using Aspergillus sp. The biogenic nanoprobe displayed marked sensing efficiency in the presence of Pb ions over concentration ranges from 5 to 100 μM with limits of detection of around 2.45 μM. Performance optimization studies revealed that the maximum fluorescence quenching efficiency was obtained in the presence of [ZnS NPs] at 4 mg/ml, and alkaline pH of 10 recorded under stable ambient temperature for approximately 5 min for the quenching process. Advanced morphological analysis indicated that the bio‐sensing mechanism was essentially a surface‐based phenomenon in which the Pb ions were in very close proximity to the QDs and formed stable ground‐state Pb–ZnS complexes, resulting in a quenched fluorescence of the QDs. Simultaneously, a larger fraction of Pb ions interacted via collisions with the excited ZnS QDs and resulted in an effective energy transfer from the excited QDs to the Pb ions, therefore resulting in an obvious decrease in QD fluorescence. These insights were well supported by theoretical analysis using Stern–Volmer plots and sphere‐of‐action models.