Methemoglobin is an oxidative form of hemoglobin in erythrocytes. The authors’ aim was to develop a new biological dosimeter based on a methemoglobin assay. Methemoglobin in peripheral blood (of females or males) that was exposed to a 60Co source (0.20 Gy min−1) was quantified using an enzyme-linked immunosorbent assay. The dose range was 0.5–8.0 Gy. In a time-course experiment, the time points 0, 0.02, 1, 2, 3, 7, 15, 21, and 30 d after 4‐Gy irradiation of heparinized peripheral blood were used. Methemoglobin levels in a lysed erythrocyte pellet from the irradiated blood of females and males increased with the increasing dose. Methemoglobin levels in female blood irradiated with γ-doses more than 4 Gy were significantly higher than those in male samples at the same doses. Two dose-response relations were fitted to the straight line: one is with the correlation coefficient of 0.98 for females, and the other is with the correlation coefficient of 0.99 for males. The lower limit of dose assessment based on methemoglobin is about 1 Gy. Methemoglobin levels in blood as a result of auto-oxidation increase after 7‐d storage at −20 °C. The upregulation of methemoglobin induced by γ-radiation persists for ∼3 d. The absorbed doses that were estimated using the two dose-response relations were close to the actual doses. The results suggest that methemoglobin can be used as a rapid and accurate biological dosimeter for early assessment of absorbed γ-dose in human blood.