Background: Body fluid balance assessment for critically ill children is vital for pediatric nurses. Aim of the present study was to evaluate the effect of educational program on pediatric nurses' knowledge and practice regarding assessment of body fluid balance for critically ill children. Subjects and Method: A quasi-experimental (pre-posttest) research design was applied at Emergency and Rehydration units of Assiut University Children Hospital. Two tools were used to collect the data: Tool (1): A structured interview questionnaire for nurses. It consisted of two parts: personal characteristics of the studied nurses and nurses' knowledge questionnaire. Tool (2): Nurses' practice for assessing body fluid balance observation checklist. Results: It was noticed that more than two-thirds of nurses had good score of knowledge after the program application. Also, the majority of nurses had a satisfactory practice level after the program application. Statistically significant differences were found between pre and post-program as regards nurses' knowledge and practice. Moreover, it was found a positive correlation regarding total nurses' knowledge and practice scores before and after program application. Conclusion: There was a significant improvement in pediatric nurses' knowledge and practice regarding assessment of body fluid balance for critically ill children after educational program implementation compared to before the program. Recommendations: Continuous educational training programs regarding assessment of body fluid balance of critically ill children will help pediatric nurses to update their knowledge and improve their practice.