The tetracarboxylate ligand H4TBAPy based on the strongly fluorescent pyrene cores and bismuth iodide constructed a permanently 3D microporous fluorescent metal–organic framework Bi-TBAPy first . In this work, the photophysical properties, quantum yield, and lifetime of the framework were studied fully. Notably, Bi-TBAPy demonstrates unique "turn on" fluorescence sensing behavior toward biothiol molecules, for example, Cys, GSH, and Hcy. The limits of detections are 1.41 μM (Cys), 1.71 μM (GSH), and 1.10 μM (Hcy), respectively. Biological activity was studied by CCK-8, flow cytometry, and fluorescence microscopy. The experimental result will hopefully be useful information when using Bi-TBAPy as a fluorescent probe.