The invention discloses a naphthenic-base mono substituted amino fluorescein compound as well as a preparation method and application thereof. The naphthenic-base mono substituted amino fluorescein compound has a structural general formula (described in the specification) or a free form thereof, wherein n is 0-9, and m is 1-9; when n is equal to m, R is mono substituted groups such as a hydrogen group, methyl, ethyl, propyl and halogen; and when n is not equal to m, R is the hydrogen group. The naphthenic-base mono substituted amino fluorescein compound can be used for detecting distribution and imaging of luciferase in vitro, cells and vivo by detecting the presence and quantity (including enzyme level, cellular level and animal level) of the luciferase by virtue of bioluminescence, can be also used for detecting distribution and imaging of ATP in vitro, cells and vivo by detecting the presence and quantity (including enzyme level, cellular level and animal level) of ATP and can be used for detecting pharmacological action of a medicine in enzyme level, cellular level and animal level as a report signal in the presence of luciferase, ATP and Mg<2+>.