The invention discloses a 2-formyl rhodamine derivative, as well as a preparation method and application thereof. The 2-formyl rhodamine derivative has a structural general formula as shown in formula(I). The preparation method comprises the following steps: reacting a rhodamine compound, ethanolamine and absolute ethyl alcohol for 8-21 hours at 75-85 DEG C, cooling to room temperature, and filtering to obtain a solid; dissolving the solid in a tetrahydrofuran solution, adding a reducing agent, stirring for 1-8 hours at room temperature, quenching with water, extracting with dichloromethane,and performing column chromatography purification to obtain the 2-formyl rhodamine derivative. According to the 2-formyl rhodamine derivative constructed by the method, formyl can be used as an activegroup and reacts with multiple substrates, and can be applied to protein labelling and other fields.