A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (Tre-Cz), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the aryl azide generating a fluorescent product through an efficient intramolecular C-H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria. Both the photoactivation and detection can be achieved using a handheld UV lamp, and bacteria of 103 CFU/mL or higher can be seen by the naked eye. The probe was also able to image mycobacteria spiked in sputum samples, although the detection sensitivity was lower. Studies using heat-killed, stationary phase and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-Cz. The uptake decreased in the presence of trehalose in a concentration dependent manner, indicating that Tre-Cz hijacks the trehalose uptake pathway. Mechanistic studies revealed that the trehalose recycling transporter LpqY-SugABC is the primary pathway for the uptake of Tre-Cz. The uptake was mostly diminished in the LpqY-SugABC deletion mutants ΔlpqY, ΔsugA and ΔsugB. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant ΔAg85C, and no incorporation of Tre-Cz into the mycomembrane was observed.