Background and Objective: Diagnosis of Hirschsprung’s disease (HD) as the most common cause of neonatal intestinal obstruction is based on the presence of aganglionosis from seromuscular or full thickness biopsy. Due to the complication of full thickness or seromuscular rectal biopsy, mucosal-sub mucosal biopsy is more intended. However, interpretation of these biopsies stained with hematoxylin and eosin (H&E) and even using immunohistochemical (IHC) methods such as acetylcholine esterase is often problematic. Although neuron-specific enolase staining (NSE) is an available and easy method to perform for diagnosis of HD, however, our knowledge on its specificity is not adequate. Therefore, this study was aimed to determine the diagnostic value of NSE on the mucosal-sub mucosal rectal biopsy for the diagnosis of HD and the allied disorders deficit. Materials and Methods: This study was conducted on 65 mucosal-submucosal and 65 seromuscular rectal biopsies (standard) obtained from the patients suspected of HD and allied disorders referred to the Avicena and Shafa hospitals (Sari, Iran) from April 2003 to September 2004. Two biopsies were taken from each patient: the mucosal-submucosal biopsy was stained by NSE and H&E staining was used for seromuscular samples. The prepared slides were observed and evaluated at double blind condition and the results were compared. Results: Sensitivity, specificity, efficiency, positive and negative predictive values in the diagnosis of HD in NSE method were 100%, 84.2%, 89.1%, 81.8%, and 100% respectively (p<0.05). On evaluation of hypoganglionosis, there were one false-negative and nine false-positive. Conclusion: In NSE staining, finding ganglion cell definitely rules out HD, but lack of ganglion cell confirms 81.8% of H.D cases. Thus, NSE staining on mucosal- submucosal specimens is possibly adequate for establishing the presence or absence of ganglion cells.