Unconventional fluorescent materials have attracted intense and continuous attention due to the facile processability, excellent biocompatibility, and high availability. However, for the lack of suitable unconventional fluorescent platform, unconventional luminophore‐based fluorescent probes have not been applied in the biological field, especially in the detection of bioactive molecules. In this work, unconventional red fluorescence is observed from a series of organoalkoxysilanes for the first time. Particularly, the unique fluorescence derived from smart Si–O bridged structures prompt the fluorescent probe design strategy. The strategy involves applying the Si–O bridge to provide desirable red unconventional fluorescence, and ratiometric detection of endogenous nitric oxide in lysosomes and in vivo. It is expected that this novel strategy will expand the applications of unconventional fluorescence to the bioimaging field, and further provide valid approach for the future evolution of unconventional fluorescent probes.