Poultry farming has the risk of being attacked by various diseases. One effort to protect the poultry, particularly layer chickens is by implementing a biosecurity system to prevent death due to diseases [1]. The area where most breeders have implemented a biosecurity system is located in Lendah, Kulonprogo because, in 2015, chickens in the district were attacked by Newcastle disease called Tetelo. This study aims to evaluate the implementation of the biosecurity system by analyzing the conditions and the success rate of this system on layer chicken farming, as well as the attitudes of breeders toward the system. The respondents were gathered using census, and the data were analyzed by measuring variables in aspects of biosecurity conditions using a rating scale. The Spearman Rank measured the correlation, whereas breeders’ attitudes toward the implementation of biosecurity were measured qualitatively. The results showed that the implementation of the biosecurity system was at a moderate level, and chicken mortality was quite high. The attitudes of the breeders toward the layer chicken’s biosecurity system as a whole belonged to the good category. In conclusion, layer chicken breeders had a positive response to the biosecurity system.