Proper biosecurity practice is crucial in poultry farming to reduce the risk of diseases. A study was conducted in Adama, Ada’a, and Lume districts, central Ethiopia, to assess the biosecurity measures of commercial poultry farms. A total of 51 farms were randomly selected, and their geographical locations were recorded using GPS devices. The biosecurity level of each farm was assessed using an observation checklist and a biosecurity score. The collected data were analyzed using Stata version 16 for binary data, and QGIS version 3.24.1 was used to map the farms. The results of the study showed that the farms were located close together within 5 km square, 3 km square, and 6 km square areas in Ada’a, Lume, and Adama, respectively. The average distance among the farms was 933.8 meters. The majority of the farms were located within 100 meters of residential areas. Only 58.8% of the farms had adequate space between sheds; 60.8% did not have an adequate drainage system. Ninety-eight percent of the farms obtain chicken from certified breeding enterprises; 92.2% provide underground water for their chickens; 80.4% report frequent cleaning and disinfection of farms; and 82.4% disclose vehicle movement control. Nearly one-third (31.4%) of the farms practice isolation of sick birds but keep them within the same shed where healthy birds are kept. The assessment of biosecurity levels of the study farms revealed that 23.5% (n=12) of the farms had a good biosecurity score, while 76.5% (n=39) had a poor score. The biosecurity scores of the farms showed a statistically significant difference among the study districts (p=0.040), with the Lume district having higher biosecurity scores than Adama and Ada’a districts. The age of the farm owners was significantly associated with the biosecurity score (p=0.003); all farms (100%) owned by individuals aged 20 - 39 had good biosecurity scores, and 80% of those farms owned by individuals aged 40 - 59 had good biosecurity. Farms owned by individuals who have previous chicken-rearing experience had better biosecurity scores than those farms owned by non-experienced owners (p<0.001). Among the farms having professional consultants, 57.7% of them had good biosecurity scores, whereas only 12.0% of farms lacking professional consultants had good biosecurity scores (p=0.015). In conclusion, the study revealed that biosecurity measures were not adequately implemented in poultry farms in central Ethiopia. Farm owners, veterinarians, and livestock authorities must collaborate to implement biosecurity measures to minimize the risk of losses and public health.