Porcine epidemic diarrhea virus (PEDV) LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province, and was identified by immunofluorescence test, immunelectronmicroscopy, RT-PCR and indirect ELISA assay. Characteristics of the virus culture and the methods of improvement of virus titer were explored. The results showed that the virus had the typical appearance of the coronavirus. Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains. Indirect immunofluorescence assay showed a significant presence of green fluorescence, and an average P/N ratio of 7.6 by indirect ELISA assay. Taken together, these tests showed positive isolation of PEDV. Using the virus plaque purification cloning methods established in the test, the purified PEDV large plaque and small plaque were obtained, and the large plaque and small plaque titers were measured with significant difference. These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.