According to the sequences of small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium spp., universal and species-specific primers were designed to detect malaria and identify species. 60 blood samples were detected by the established nested PCR method. The results were compared with those of microscopic examination. 40 blood samples were Plasmodium-positive by nested PCR with 22 samples of P. falciparum, 13 of P. vivas, 3 with P. falciparum and P. vivax mixed infection, 1 of P. ovale and 1 of unclassified malaria infection. Altogether, the coincidence between the results of nested PCR and microscopy stood for 76.7% (46/60), including 18 of P. falciparum, 11 of P. vivax and 17 negatives. Further sequence analysis and real-time PCR were performed to detect blood samples with discrepancy, results of which were the same as that of nested PCR. The amplified product of P. ovale was sequenced and showed 100% homology to the corresponding part of P. ovale SSU rRNA gene sequence (GenBank No. DQ845247), which confirmed that the case was imported ovale malaria.