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Simple Summary: Evaluation of biosecurity measures remains essential for successful control and prevention of diseases in a country like Ethiopia, where the poultry sector is flourishing. This research was conducted from October 2020 to June 2022 and aimed to assess the level of implementation of biosecurity practices of 226 poultry farms belonging to different scales found in three selected areas of central Ethiopia, including Addis Ababa, Bishoftu and West of Shaggar City. The study showed a very low overall biosecurity practices score compared to the global average. All three study areas had rather similar levels of biosecurity practices, being statistically insignificant. The majority of the external biosecurity components were highly disrupted and far below the global average. Similarly, the internal biosecurity practices were found to be still lower than the global average. Thus, weak and relaxed biosecurity practices were evident among poultry farms in central Ethiopia. Understanding the poor status of the implementation of appropriate biosecurity practices will help to design and develop strategies that safeguard the poultry production sector from the impact of various deadly diseases. The findings emphasize the need for active involvement of all stakeholders in the poultry production value chain towards boosting the productivity of the sector in Ethiopia. The present study aims to assess the level of implementation of biosecurity practices of small-, medium- and large-scale poultry farms in central Ethiopia. A cross-sectional study design was implemented involving 226 poultry farms (153 small-, 53 medium- and 20 large-scale farms) in three selected areas of central Ethiopia, including Addis Ababa, Bishoftu and West of Shaggar. The results revealed a very low overall biosecurity score of 43.1% for central Ethiopia compared to the global average score of 64.3%. No significant difference (p > 0.05) in the overall biosecurity practices among the three study sites (36.1% for Addis Ababa, 49% for Bishoftu and 34.9% for West of Shaggar) was observed. Six of the eight external biosecurity components with an overall score of 40.7% as compared to the global average of 64.6% were highly disrupted and scored lower values. With regard to internal biosecurity disease management, cleaning and disinfection practices were found to be 52.6% as opposed to the global average of 64%. The poor biosecurity level among poultry farms of central Ethiopia clearly demonstrates the urgent need for the implementation of appropriate biosecurity practices through the active involvement of all stakeholders to combat the impact of various diseases and boost the productivity of the sector. [ABSTRACT FROM AUTHOR]