Indirect Fluorescent-Antibody Test (IFA), Western Blot (WB) and Nested-PCR were applied to identify the Borrelia burgdorferi in human serum samples in Hainan province. A total of 259 serum samples were collected from Sanya Peoples' Hospital, Hainan province. These samples were examined for the presence of B. burgdorferi serologically and etiologically by the two tier tests (IFA and WB) and Nested-PCR. 43 in total of 259 serum samples were tested positive by IFA assay, the positive rate was 16.6%. Among 43 IFA-positive samples, 6 were identified positive by WB. Nested-PCR were also used to test B. burgdorferi DNA in 259 serum samples at the same time, 27 samples were tested positive with positive rate of 10.42%. It is the first time to confirm that there are Lyme patients in Hainan province of China. The study suggested that Lyme disease should be commonly considered by clinicians with the patients who had correlated symptoms with lyme disease in Hainan.