Background — Hepatitis B is a widespread infectious disease throughout the world. Clinical and laboratory diagnosis of this infection is important. Rapid test of hepatitis B, known as cassette test, is a useful test that is easy to perform as well as it may have lack of sensitivity and specificity. Materials and Methods — Sera of a total of 55 patients were used for the study. All the sera that were tested for hepatitis B surface antigen (HBsAg) using macro enzyme-linked immunosorbent assay (Macro ELISA) were stored at -20°C for a maximum period of 3.5 years. The sera were thawed and tested again for HBsAg using the Nanosign rapid test kit (Bioland, South Korea) for hepatitis B virus (HBV) infection. Results — A total of 16 (29.1%) sera revealed negative and the rest 39 were positive, meaning of a positivity rate of 70.9%. Rapid test revealed a very low positivity rate of 6.6% in sera with below 100 IU/ml level of HBsAg. In contrast, only two of the rest 40 sera with HBsAg levels above 100 units were negative, meaning of a positivity rate of 95.0% in high HBsAg level samples. A highly significant positive correlation was found between positivity levels and HBsAg levels (R=0.734, P<0.001, correlation is significant at the 0.01 level by 2-tailed analysis). We didn’t find any correlation between positivity and the period passed after freezing. Besides this, no significant difference was observed amongst the groups in terms of the time intervals of freezing. These findings suggest that the time passed after the first freeze of the sera didn’t cause any impact on the results of rapid tests. Conclusion — The findings of the present study suggest that the rapid test of HBV infection is reliable in sera with high levels of HBsAg, and the time passed after freezing of the sample doesn’t change the results of the tests. Besides this, a negative rapid test result doesn’t rule out the infection.