The great success of a His₆-Ni-nitrilotriaceate (Ni-NTA) system in protein purification has inspired scientists to develop novel Ni-NTA based fluoresent probes for imaging of proteins in live cells. Despite a great deal of effort being made, only very few such probes can cross the cell membrane to label intracellular proteins. Herein, a new red fluorescence probe (Ni-NTA-AB) consisting of a Ni-NTA moiety, BODIPY and an arylazide was designed and synthesized to selectively label His₆-tagged proteins. Guided by the Ni-NTA, the probe selectively binds to proteins with a genetically fused His₆-tag exemplified by a DNA repair protein, xerodermapigmentosum group A (XPA122), leading to ca. 26-fold fluorescence enhancement. Ni-NTA-AB could enter into live bacterial cells to label intracellular His₆-tagged proteins with the assistance of Tween 80 and also to quantify His₆-tagged proteins in SDS-PAGE upon photoactivation of arylazide with a wide linear range (25–1000 ng). This study may provide a new horizon for the multiple-channel tracking and labelling of His₆-tagged proteins.