Background: Brucellosis is a zoonotic disease with different clinical symptoms. Its early diagnosis is essential to prevent severe complications. Due to the limitations of serological diagnostic methods, the polymerase chain reaction (PCR) method has become important in the diagnosis of the disease. Objectives: Our study aimed to evaluate the PCR method in patients with suspected brucellosis and compare it with serological tests. Methods: This cross-sectional study was performed on 90 febrile patients with clinical features of brucellosis who were examined by an infectious disease specialist. A total of 90 serum samples were collected from the suspected brucellosis patients admitted to the hospital and were analyzed by serological (Rose Bengal) and PCR tests. Then, each method's results were recorded and compared with each other. Results: According to serological test results, 45 samples were negative, and 45 were positive. Then, among the serology-positive patients, all had positive PCR results. However, 40 out of 45 patients had a positive PCR test in serology-negative patients. According to this study, the sensitivity of PCR in diagnosing human brucellosis with the serology-positive test is 100%, and with the negative serology test is 88.9%. Therefore, the sensitivity of PCR is higher than that of serology tests in patients, which was 50% in this study. Conclusions: The PCR test can be a valuable diagnostic method for patients with negative serologic test results.