Background: Biosecurity in the poultry refers to a set of practices and measures taken to limit, control, or prevent the introduction and dissemination of infectious diseases in the poultry premises and facilities. Material and Methods: A survey was employed from January 2020 to October 2020 to assess the knowledge and practices of the biosecurity status of 36 commercial chicken farms established in Dire Dawa, Eastern Ethiopia, by interviewing farm owners using a structured questionnaire. The gathered data was summarized using frequency tables and analyzed with Pearson’s chi-square test and Fischer’s exact value using Stata 14 statistical software. Results and Discussion: From the assessed chicken farms, 25 (69.44%) had previous experience in rearing chickens. Among those owners, 9 (25%) refused to disclose their level of education, and 16 (44.44%) didn’t receive training on chicken farm management. Among the 36 Dire Dawa chicken farms, 24 (66.67%) were located within 0-50 m from the main road and 29 (80.56%) farms were established within 500 m from the nearest farm and 21 (58.33%) placed within 0-20 m from residential areas. (Table 3). 30 (83.33%) participants disclosed their employees didn’t receive training on biosecurity. From the chicken farms assessed, 26 (72.22%) had fences, 32(88.89%) had footbaths at the gate, 27 (75.00%) prohibited the entrance of visitors, 3 (8.33%) didn’t exchange equipment with other farms, each of the 36 (100%) farms didn’t used surface water for drinking or cleaning, 20 (55.56%) stay informed regarding disease outbreak in the area (Table 4). However, only 5 (13.89%) undertook a permanent rodent control strategy. Furthermore, 22 (61.11%) farms were easily accessed by wild birds, each of the 34 (94.44%) farms purchased day-old chicks, feed and shared trucks as well. Among the assessed farms, only 2 (5.56%) had signages to restrict people's access, 7 (19.44%) had isolation room for diseased chickens, 11 (30.56%) farm properly ......