A highly selective and red emissive fluorescent probe (HCyNB) for penicillin G amidase (PGA) has been prepared in a simple two-step process. After enzymatic reaction, HCyNB showed a fast response to PGA and afforded a light-up red fluorescence assay of PGA with a detection limit of 0.067 U L⁻¹, without interference from other biologically-relevant species and enzymes, including ROS. The easy synthesis, fast response, good sensitivity, remarkable selectivity and pH independence render HCyNB an outstanding tool for bioimaging of endogenous PGA in penicillinase-producing Bacillus subtilis.