Objective To prepare the anti-herpes simplex virus-1 （HSV-1） egg yolk antibody （IgY） and discuss its biological activity， and to clarify its anti-HSV-1 capability. Methods The inactivated HSV-1 vaccine was prepared， the high egg-producing hens were immunized by multi-site injection method in the chicken breast， and IgY was purified by polyethylene glycol-6000 （PEG-6000） method；the titer， purity， and protein levels of IgY were detected by indirect ELISA method， sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） technology， and protein concentration quantification kits； the ability of IgY to neutralize the virus was detected by indirect ELISA method； the virus blocking rate was detected.The Vero cells were used as the target cells for viral infection. The cytopathic effect （CPE） at different antibody concentrations （0.015 60， 0.031 25， 0.062 50， 0.125 00， 0.250 00， 0.500 00， and 1.000 00 g·L?1） was detected；the in vitro anti-HSV-1 capacity of IgY was detected according to the degrees of CPE. Results The titer of the prepared IgY was gradually increased with the extension of immunization time. The titer of the IgY stabilized at 1/1 024 000； the clear bands for the IgY light and heavy chains were observed； the average protein level of IgY was 11.544 g·L?1； the blocking rate of IgY against HSV-1 could reach up to 77.90%； the degree of HSV-1 induced CPE was decreased with the increasing of the antibody concentrations， and when the concentration of IgY reached 0.500 00 g·L?1 or higher， less than 25% of the cells （even no cells） exhibited cytopathic changes. Conclusion Anti-HSV-1 IgY is successfully prepared， and this antibody has significant neutralizing and blocking capabilities and in vitro inhibitory activity against HSV-1， which can be utilized for the development of drugs and diagnostic methods.