Objective To compare different blocking methods for eliminating autofluorescence of mouse liver frozen sections, so as to find the best method to reduce the interference to immunofluorescence positive signals and improve the accuracy of immunofluorescence. Methods Intrasplenic injection-liver colonization nude mice:Hepa1-6-GFP cells were intrasplenically injected into male athymic BALB/c nude mice to create liver colonization models. Liver tissues were frozen and continuously sectioned. Sections were blocked with AB reagent (A reagent:streptavidin reagent, B reagent:biotin reagent), blocking buffer, AB reagent+blocking buffer, or acetone+AB reagent+blocking buffer, and then the autofluorescence of the frozen sections was detected. C57BL/6 mice:the liver tissues of C57BL/6 mice were frozen and continuously sectioned, and then the sections were blocked with AB reagent, blocking buffer, AB reagent+blocking buffer, or acetone+AB reagent+ blocking buffer. Liver macrophages (Kupffer cells) were labeled with F4/80, and then the autofluorescence of mouse liver frozen sections was detected. Results In the immunofluorescence staining of liver tissue frozen sections, all the above four blocking methods could reduce the autofluorescence of liver sections, and acetone+AB reagent+blocking buffer group had the best effect. Conclusion The combined buffers (acetone+AB reagent+blocking buffer) has the best effect in eliminating the autofluorescence of mouse liver frozen section.