Abstract As a first-line tuberculostatic drug, isoniazid (INH) plays effective and irreplaceable role in prevention and treatment of tuberculosis. In this work, a rapid and simple signal-on fluorescence approach is established for INH assay by employing a platform composed of silver nanoclusters (AgNCs) and MnO2 nanosheets. In the proposed sensing system, strong red fluorescence of poly (methacrylic acid)-stabilized AgNCs can be greatly quenched after they attach to the surfaces of MnO2 nanosheets. With the addition of INH, MnO2 nanosheets are reduced to Mn2+ and subsequently release the AgNCs, which leads to obvious fluorescence recovery again. Based on this mechanism, highly sensitive detection of INH in the range of 0.8–200 μM is realized (detection limit: 476 nM). The present strategy shows remarkable advantages including simplicity, rapidness, high sensitivity and wide detectable range. This method is also practical and comparable to high-performance liquid chromatography, which can be applied to detect INH in human urine and serum samples as well as pharmaceutical products.