To clarify the biological and physicochemical properties of Anguillid herpesvirus (AngHV), an AngHV strain, NA16108, isolated from the diseased Anguilla anguilla of "mucus sloughing and hemorrhagic septicemia disease" was used to study its replication characteristics and infection sensitivity to some fish continuous cell lines, and its survivability to heat, acid, alkali, chloroform, ether and other physical and chemical factors was further analyzed. The results indicated that AngHV infected eel ovary cells (EO) showed typical sequential cytopathic effects (CPE), and many typical herpesvirus-like viral particles could be seen in the infected cells. AngHV could propagate stably in EO cells, and the suitable temperature for propagation is 25 °C to 27 °C, but it could not propagate in epithelioma papilloma cyprinid cells (EPC), grass carp ovary cells (CO), fathead minnow cells (FHM), chinook salmon embryo cells (CHSE-214), rainbow trout gonad cells (RTG-2), bluegill fry cells (BF-2). The titer of AngHV slightly decreased after 30 min treatment at 37 °C, and it could be completely inactivated after 30 min treatment at 56 °C. AngHV is sensitive to acid (pH 3.0), but resistant to alkali (pH 10.0), and highly sensitive to chloroform, ether and other organic solvents. These results can provide references for the prevention and control of AngHV diseases.