The invention discloses a malononitrile derivative near-infrared hydrogen sulfide fluorescent probe as well as a preparation method and application thereof. The probe has a structure represented by aformula I shown in the description. The preparation method comprises the following steps: firstly, mixing isophorone, malononitrile and piperidine, performing heating reflux under the protection of aninert gas, performing purification, mixing the obtained purified product, p-hydroxybenzaldehyde and piperidine, performing heating reflux under the protection of an inert gas, performing purification, mixing the obtained purified product, urotropine and trifluoroacetic acid, performing heating reflux under the protection of an inert gas, performing purification, finally mixing the obtained purified product, 2,4-dinitrobenzenesulfonyl chloride and triethylamine, performing a reaction under stirring at room temperature, and performing purification to obtain the fluorescent probe. The fluorescent probe provided by the invention has large Stokes shift (229 nm), a longer fluorescence emission wavelength (653 nm) and high sensitivity, can be used for specific detection of biological hydrogen sulfide, and has good biofilm permeability and low cytotoxicity; and the fluorescent probe has a simple synthetic route, a high yield and large practical values.