A fluorescent sensor based on guanidinium-tethered tetraphenylethene (TPE) has been investigated toward the differentiation of pyridine nucleotide cofactors (NAD+, NADH, NADP+, and NADPH). TPE selectively recognizes NADPH possessing the higher tetra-anionic net-charge, resulting in the steep turn-on fluorescence increase. The comparative aggregation behaviors and fluorescence response studies of TPE on the four cofactors reveal that the critical aggregate concentration of TPE against NADPH correlates directly with the concentration threshold for the fluorescence response. These results establish that TPE can selectively differentiate NADPH over the other three cofactors by the steep aggregation-induced fluorescence response accompanied by the high signal-to-background contrast.