Aim: A comparative study of immunohistochemical, hematoxylin & eosin staining and its diagnostic importance in Hirschsprung’s disease. Material and Methods: The study of 510 patients comprised colorectal, appendicectomy biopsies and myectomy specimens at various levels. The study included both ganglionic and aganglionic segments of intestine. The specimens were fixed in 10% formalin solution. In the laboratory, the sections of paraffin embedded tissues were stained H & E and compared with Cathepsin D; repeated sections were taken from these cases for the demonstration of H & E and Cathepsin D. Results: In our study of 357 cases, 223 are male children and 74 are female children (Male: Female ratio-3:1). Short segment was the most commonly occurring type constituting 229 cases (64%), while long segment was 77 cases (21.5%). The less common is the total colonic aganglionosis constituting 21 cases (5.8%). Conclusions: Cathepsin D is equally good like Acetyl cholinesterase and can be used as a reliable immune-histo chemical stain in detecting immature ganglion cells..