Abstract: Bioconjugation techniques involve the covalent attachment of synthetic labels to biomolecular frameworks and have been extended to the labeling of biomolecules in vitro and in vivo. Fluorescence labeling techniques that covalently attach a colorful fluorescence tag to biomolecules allows for more exciting life science research opportunities. In this paper, we described a novel immunofluorescence labeling technique based on click chemistry. Two key compounds 2,5-dioxopyrrolidin-1-yl 6-azidohexanoate (compound 1) and 4-ethynyl-N-ethyl-1,8-naphthalimide (compound 2) were synthesized first. Compound 1, which has an active succinimide residue, can connect with primary amine groups (-NH2) in the side chain of lysine (K) residues of IgG antibodies. Compound 2 and the specific substrate azide were used to induce a fluorescence group, via copper (I)-catalyzed 1,2,3-triazole, to generate a reaction between azides and the terminal alkynes. Compared with NHS-FITC- or NHS-rhodamine-labeled IgG staining, the azide-labeled IgG showed the same sensitivity and specificity. Furthermore, this labeling method was successful in antibody conjunction. Three-channel fluorescent cross imaging analysis was localized to three tumor-associated antigens, including Her2, GRP94 and EFGR4, and was performed by laser-scanning confocal microscopy. We found that the immunofluorescence cross-imaging enabled visualization of the FITC-labeled secondary antibody, the biotinylated antibody with Cy3-labeled streptavidin, and click chemistry fluorescence staining for azide-labeled IgG. The fluorescent staining method based on click chemistry is a novel immunofluorescence technique that offers an important technique for immunofluorescence analysis with potential for a wide range of applications in future immunostaining research.