The unreasonable of mortality rate of the layer poultry farm in Village Gulurejo still become a main problem. Although breeders claimed they have applied biosecurity system in their layer poultry farm, but the unreasonable of mortality rate still could not be fixed. This studi aims to know the condition level of biosecurity and level of succes aplicatation in layer poultry farm in Village Gulurejo. The condition refers from nine aspects which are sanitation of the poultry coop and surroundings, sanitation of drinking place, sanitation of food place, vaccinations, handling of poultry’s feces, handling of sick poultry, handling of dead poultry, quarantine, traffic control in poultry coop. This study has done in Village Gulurejo Subdistrict Lendah Regency Kulonprogo. Census method was selected that means all of poultry farmers were involved in this study. Collecting datas have done by interviewed and observation directly to every poultry farmer and poultry coops. The analysis technique used rating scale and spearman correlation. The result of this study showed that the biosecurity level of poultry farming in Village Gulurejo was in medium level. That condition is caused by farmer’s less attention to biosecurity system especially in sanitation of food place and traffic control aspects. In the other hand, most of farmers did practice well such as in vaccinations, handling dead poultry and quarantine. In the other hand, for the level of succes aplication, as many as 82% farmers were not succes yet in biosecurity aplication because the mortality level is still high. The rest farmers which are 18% are succes aplicated the biosecurity because of the reasonable mortality level. Key words: biosecurity level, layer poultry farming, Village Gulurejo