The conveniently obtained water-soluble small-molecule L1-5 were selective for RNA with remarkable "off-on'' two-photon fluorescence responses in vitro and in vivo, showing that L1-5 can surprisingly stain mitochondria or nucleoli individually with orange fluorescence under different focal lengths. Compounds L6-8 were prepared, for comparison, to further explore the mechanism of the binding of L1-5 with RNA, which revealed that the synergistic effect of the amino group and the pyridinium cation played a significant role in distinct cell imaging.