The invention relates to a pyrrole-rhodamine acylhydrazone derivative as well as a preparation method and application of the derivative, belonging to the organic synthesis field. The preparation method of the pyrrole-rhodamine acylhydrazone derivative comprises the following steps: (S1) adding N-(2-morpholin-4-ethyl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide into an organic solvent, and adding rhodamine 6G hydrazide, so as to obtain a mixture; (S2) carrying out reflux stirring reaction on the mixture obtained in the step (S1) at a normal pressure; (S3) after the reaction is finished, cooling to the room temperature so as to separate solids, carrying out reduced pressure filtration, and extracting filter residue; and (S4) washing the filter residue obtained in the step (S3), so as to obtain the pyrrole-rhodamine acylhydrazone derivative. The pyrrole-rhodamine acylhydrazone derivative is capable of selectively acting with bivalent copper ions, blue fluorescence is changed into greenfluorescence, a ratio fluorescence effect is realized, the naked eye distinguishing detection can be realized, and particularly, the pyrrole-rhodamine acylhydrazone derivative can be taken as a fluorescence probe to be applied to the convenient detection of bivalent copper ions in cell lyase.