Background The Airway, Breathing, Circulation, Disability and Exposure (ABCDE) approach is a universal, priority-based approach for the assessment and treatment of critically ill patients. Although the ABCDE approach is widely recommended, adherence in practice appears to be suboptimal. The cause of this non-compliance is unknown. As knowledge is a prerequisite for adherence, the aim of this study was to assess healthcare professionals’ knowledge of the ABCDE approach. Methods A cross-sectional study was conducted at the Radboud University Medical Center, the Netherlands. A digital multiple-choice assessment tool of the ABCDE approach was developed by an expert panel through a mini-Delphi method and validated by performing test item statistics and an expert-novice comparison. The validated test was sent to healthcare professionals (nurses, residents and medical specialists) of the participating departments: Anaesthesiology, Paediatrics, Emergency Department and the Neonatal, Paediatric and Adult Intensive Care Units. Primary outcome was the test score, reflecting individual level of knowledge. Descriptive statistics, regression analysis and ANOVA were used. Results Test validation showed a Cronbach’s alpha of 0.71 and an expert-novice comparison of 91.9% (standard deviation (SD) 9.1) and 72.4% (15.2) respectively (p < 0.001). Of 954 eligible participants, 240 filled out the questionnaire. The mean (SD) test score (% of correct answers) was 80.1% (12.2). Nurses had significantly lower scores (74.9% (10.9)) than residents (92.3% (7.5)) and medical specialists (88.0% (8.6)) (p < 0.001). The Neonatal Intensive Care Unit (75.9% (12.6)) and Adult Intensive Care Unit (77.4% (11.2)) had significantly lower scores than Paediatric Intensive Care Unit (85.6% (10.6)), Emergency Department (85.5% (10.4)) and Anaesthesiology (85.3% (10.6)) (p < 0.05). Younger participants scored higher than older participants (−0.30% (-0.46;-0.15) in test score/year increase in age). Conclusion Scores of a validated knowledge test regarding the ABCDE approach vary among healthcare professionals caring for critically ill patients. Type of department, profession category and age had a significant influence on the test score. Further research should relate theoretical knowledge level to clinical practice. Tailored interventions to increase ABCDE-related knowledge are recommended.