A multi-branched fluorogenic probe for the rapid and specific detection of Gram-negative bacteria is reported. Three Gram-negative-targeting azido-modified polymyxins were clicked onto a trivalent scaffold functionalised with the environmental green-emitting fluorophore 7-nitrobenz-2-oxa-1,3-diazole. The probe allowed wash-free detection of target bacteria with increased sensitivity and lower limits of detection compared to monovalent probes.