A photoinduced electron transfer (PET) and chelation-enhanced fluorescence (CHEF) regulated rhodamine-azobenzene chemosensor (L) was synthesized for chemoselective detection of Al, Cr, and Cu by UV-Visible absorption study whereas Al and Cr by fluorimetric study in EtOH-HO solvent. L showed a clear fluorescence emission enhancement of 21 and 16 fold upon addition of Al and Cr due to the 1:1 host-guest complexation, respectively. This is first report on rhodamine-azobenzene based Cr chemosensor. The complex formation, restricted imine isomerization, inhibition of PET (photo-induced electron transfer) process with the concomitant opening of the spirolactam ring induced a turn-on fluorescence response. The higher binding constants 6.7 × 10 M and 3.8 × 10 M for Al and Cr, respectively and lower detection limits 1 × 10 M and 2 × 10 M for Al and Cr, respectively in a buffered solution with high reversible nature describes the potential of L as an effective tool for detecting Al and Cr in a biological system with higher intracellular resolution. Finally, L was used to map the intracellular concentration of Al and Cr in human lymphocyte cells (HLCs) at physiological pH very effectively. Altogether, our findings will pave the way for designing new chemosensors for multiple analytes and those chemosensors will be effective for cell imaging study.