The unreasonable mortality rate of poultry in Gulurejo Village has become a major problem. Although breeders claimed they have applied Biosecurity system, the issue of poultry mortality still could not be resolved. This study aims to analyze the condition and success level of Biosecurity application in a poultry farm in GulurejoVillage. The condition refers to nine aspects which are sanitation of the poultry coops and surroundings, drinking and food place, vaccinations, handling of poultry feces, sick and dead poultry, quarantine, and traffic control in poultry coops. This study was done in Gulurejo Village, Lendah Subdistrict, Kulonprogo Regency. The census's method was selected. The analysis technique used a rating scale and Spearman correlation. The result of this study showed that the Biosecurity level of poultry farming was on a medium level. That condition was caused by farmers who paid little attention to Biosecurity system, especially in the sanitation of food place and traffic control aspects. On the other hand, farmers who applied Biosecurity system well paid adequate attention to vaccinations, handling of dead poultry and quarantine. With regard to the level of successful applications, 82% of farmers were not successful in Biosecurity application because the mortality level was still high.