Purpose: This study aims to investigate risk factors of BE, ARM and acetylcholinesterase staining related to failure of treatments and diagnostic value of Hirschsprung’s disease (HSCR) or intractable constipation (IC). Methods: A single-center retrospective cohort study of children with constipation was conducted. All patients were followed up until significant improvement of defecation. Patients initially received general/conservative treatment or surgery. Upgraded treatments were adopted if the initial treatment failed. The risk factors including transitional zone, barium residue, absence of recto-anal inhibitory reflex (RAIR) and acetylcholinesterase staining. A p value <0.1 or <0.05 was considered to be statistically significant in Logistic regression. Chi-square test was used to calculate the sensitivity and specificity when analyzing the diagnostic value.Results: This study enrolled 4869 cases. Acetylcholinesterase staining +++ had the highest risk (OR, 177.613[95%CI 92.178~273.526]) leading to failure of general treatment, followed by absence of RAIR ((OR, 158.786[95%CI 92.178~273.526]) and severe barium residue (OR, 153.829[70.004~338.028]). The leading risk factors causing failure of conservative therapy were severe barium residue (OR, 15.530[95%CI 5.337~45.191]) and acetylcholinesterase staining +++ (OR, 13.242[6.657~26.337]). In children ≤3 years, the transitional zone, absence of RAIR and acetylcholinesterase staining ++/+++ had similar specificity (80.0%, 75.6% and 73.3%), whereas the acetylcholinesterase staining ++/+++ had the highest sensitivity (95.5%). The sensitivity was generally lower in children > 3 years, however the specificity was similar between each age group.Conclusion: This real-world study validates that the performance of 3 examinations is of great significance in guiding upgrading treatments, predicting clinical outcomes and identifying HSCR and IC.Trial Number: NCT02216994 (registered on October 27, 2014)