Background: Consumers’ health would be at risk unless food preparation and handling hygiene is ensured by food establishments. Food-borne illness is a major public health problem worldwide. The supply of safe and healthy food is crucial to prevent food-borne illness. However, evidence regarding food safety knowledge and handling practice is limited in Ethiopia Especially in-flight catering industry. Objective: This study aimed to assess food safety knowledge, handling practice, and its associated factors among food handlers in Ethiopia Airlines in-flight catering. Method: The study was conducted cross-sectional from September to June 2023 in Ethiopian Airlines' in-flight catering division. A total of 277 food handlers were recruited through a simple random selection method. A structured questionnaire was used to collect the required data. SPSS version 25 was employed to analyse the collected data, and both descriptive and inferential data analysis tests were used. Frequency and percentile were used to summarize the data and test the prevalence of food safety knowledge, attitude, and practice. Chi-square test and bivariate logistic regression tests were also used to identify the significant candidate variables for the final model. Finally, multivariate logistic regression analysis was used to determine the factors significantly associated with the food handling practice of food handlers. Result: In this study, the majority—77.2% 64.6%, and 75.1%—had a good level of knowledge, a positive attitude, and a good level of practice regarding food safety, respectively. In multivariate binary logistic regression analysis, age (30-40) [AOR = 2.65, 95% CI: (1.15, 6.14)], and (above 40) [AOR = 4, 95% CI: (1.26, 7.26)] sex (male) [AOR = 0.017, 95% CI: (0.003, 0.82)], educational status (college level and above) [AOR = 3.03, 95% CI: (1.64, 6.45)], good knowledge [AOR = 5.5, 95% CI: (1.19, 9.2)], and positive attitude [AOR = 4.74, 95% CI: (2.42, 9.29)] became significant factors for food safety practice among food handlers. Conclusion: This study demonstrated that food handlers had high levels of knowledge, attitudes, and practices regarding food safety and hygiene and that these practices were significantly associated with age, sex, educational status, food safety knowledge, and attitude. Therefore, strategies targeting these factors should be considered when the catering service design strategies to improve food hygiene practices, is also better to provide additional training for younger, males, less educated food handlers to improve their skills. Keywords: Food safety practice, food handlers, knowledge, attitude, Ethiopian Airlines Catering Services