Hemoglobin is a common hematological parameter assessed in laboratory and also an important tool for clinical management of patients. Reference values being used in most laboratories in our country, have been provided from the established reference values of developed countries. Hemoglobin concentration varies usually due to age, sex, attitude, ethnic origin, dietary habits, socioeconomic status and environmental factor. Therefore, there is a need to establish reference value of hemoglobin for healthy Bangladeshi population. This study was carried out to establish the reference value of hemoglobin of healthy adult population of Dhaka, Bangladesh. This cross sectional study was conducted in the department of Physiology, Dhaka Medical College, Dhaka, Bangladesh from July 2017 to June 2018. A total number of 500 healthy subjects from different areas of Dhaka city were selected on the basis of inclusion and exclusion criteria. The subject was interviewed and detailed history was taken and recorded in a prefixed questionnaire. Hemoglobin concentration was estimated in the Department of Laboratory Medicine, Dhaka Medical College hospital, Dhaka. For statistical analysis, Unpaired Student's 't' test was performed by using SPSS windows version 19.0. The reference values (2.5th percentile- 95th percentile) of hemoglobin concentration for male and female were 10.9-15.9 and 9.4-14.7gm/dl respectively. The hemoglobin concentration was significantly higher in male than female (p<0.05). There was no statistically significant correlation of hemoglobin concentration with age of study subjects. The reference value of hemoglobin obtained from this study is lower than practiced reference value and supports the need for nationwide study to establish our population specific hematological parameters.