To establish an ideal method for the detailed definition of the tropism of the goose adenovirus new-type gosling viral enteritis virus (NGVEV) in conventional paraformaldehyde-fixed paraffin-embedded gosling tissue sections, an indirect immunofluorescence assay was established and optimized in this study for the first time. Three-day-old goslings orally inoculated with the CN strain of NGVEV were killed from 0.5 h to 30 days post-infection (p.i.) at intervals, and organs were collected and prepared for immunofluorescence assay and ultrastructural observation. NGVEV antigens could be detected in the gastrointestinal tract and lymphoid organs as early as 24 h p.i. The lymphoid organs (bursa of Fabricius, thymus, Harderian gland and spleen), the digestive organs (proventriculus, gizzard, duodenum, jejunum, ileum, caecum, rectum and liver), kidney, myocardium and brain of NGVEV-infected goslings showed extensive evidence of presence of viral antigens. Lymphocytes, macrophages, mesenchymes, endothelial cells, epithelia, superficial and crypt mucosal cells, glandular cells, and fibrocytes served as the principal site for NGVEV localization. The number of positive cells and intensity of fluorescence increased until 15 days p.i. Ultrastructural observation showed that NGVEV particles could be seen from 3 days p.i. onwards.