Activity-based near-infrared (NIR) fluorescent probes provide powerful tools for diagnosis of diseases. However, most of these probes suffer from low specificity due to "off-target" reaction. The dual-locked strategy, which utilizes two biomarkers as triggers, can increase the specificity and precision of diagnosis. Here, we report a dual-locked NIR probe, MB-m-borate, which releases fluorophore methylene blue (MB) after hydrogen peroxide-tyrosinase (H2O2-TYR) cascade activation. Both MB-m-borate and its intermediate MB-m-phenol (the product after H2O2 activation) show almost nondetectable fluorescence. MB-m-borate exhibits "turn on" fluorescence upon H2O2-TYR cascade activation. The further live cell bioimaging results indicate that MB-m-borate only responds to melanoma cells, providing it as a robust probe for precise detection of melanoma. Finally, the probe is applied for the diagnosis of melanoma in vivo with a xenogeneic mouse model.