Reference values derived from developed countries are used in many countries in Africa for interpretation of laboratory results obtained during routine healthcare and clinical trials. Use of locally derived reference values has been recommended. The purpose of the study was to establish age- and sex-specific reference values for children in the middle belt of Ghana. Reference values were determined for 21 biochemical and 18 hematologic parameters by using Clinical and Laboratory Standards Institute C28-A3 guidelines in a sample of 1,442 healthy children. Hemoglobin, hematocrit, mean cell volume, erythrocytes, urea, and creatinine were lower when compared with values from northern countries but alanine aminotransferase, aspartate aminotransferase, and total bilirubin were higher. A panel of locally relevant age- and sex-specific reference values was established for commonly used biochemical and hematologic tests in children in the middle part of Ghana. This will help in interpretation of laboratory results for clinical management of patients, screening, and safety monitoring during clinical trials.