Abstract: By using SPF chicken embryo and vero cell inoculation， a virus strain was isolated from liver and ovarian of the diseased layers that experienced egg drop， follicular atrophy and multiple organ hemorrhage in layer flock located in Jiangsu. The isolated virus can not agglutinate chicken and goose RBC and was sensitive to chloroform and ether. The viral nucleic acid was RNA. PCR screening indicated that the isolate was negative to avian influenza virus， New Castle Disease virus， egg\|laying reduction syndrome virus， infectious bronchitis virus， infectious laryngotracheitis virus, but positive to Tembusu virus. The sequence analysis showed that it had more than 99% homology with the Tembusu virus E gene published in GenBank and preserved in our laboratory. The results indicated that the isolated virus （SN01） was a kind of new Tembusu virus within the family of Flaviviridae， which was temporarily called chicken\|derived Tembusu virus.