The invention provides a berberine affinity labeling probe and a preparation method thereof and belongs to the field of biological detection. The probe comprises four functional parts including an active group, namely, berberine, an affinity labeling group, a connection part and a reporter group. After the berberine labeling probe interacts with target protein, the affinity labeling group is decomposed through illumination, an active intermediate is obtained, accordingly, probe molecules and protein at target sites are subjected to covalent binding, and target protein is called out and determined. The probe can be used for study of a target after berberine interacts with a body, tissue, cells and thalli. The berberine affinity labeling probe is based on the action between active micromolecules and target protein and has the characteristics of being convenient, accurate, high in flexibility and applicable to non-model organisms, and the sensitivity is remarkably higher than that of a protein spectrum probe and RNA (ribonucleic acid) interference. The berberine affinity labeling probe can be used for fishing target protein in human bodies, experimental animals and microorganisms and has important biological and medical application value.