Objective To establish a simple and fast paraffin section technique and staining method suitable for use with three-dimensional (3D) cell culture models and to provide a convenient pathological technology platform for the study of the morphology, function, and application of 3D cell culture models. Methods HepG2 cells or/ and LX-2 cells were cultured into 3D multicellular spheroids using an ultra-low attachment surface culture plate, and paraffin sections were made. Hematoxylin and eosin ( HE) staining, immunohistochemical staining, and immunofluorescence staining were performed to detect the histological and morphological characteristics and protein expression of the multicellular spheroids. Results A rapid paraffin sectioning method suitable for 3D cell culture models was established. The prepared sectioned cell spheroids had complete structure, clear cell boundaries, and good antigen activity. The morphological structure and the expression and distribution of the proteins of 3D cell culture models could be accurately detected by HE staining, immunohistochemical staining, and immunofluorescence staining. Conclusions The establishment of a rapid paraffin sectioning method has enriched the research method available for 3D cell culture models and could promote their application in basic research, translational medicine, and drug development.