Background: Anaphylaxis is defined as a severe, life-threatening systemic hypersensitivity reaction. Healthcare professionals must recognize the symptoms, apply correct treatment immediately, and provide epinephrine auto-injectors (EAI) to patients who experience anaphylaxis. Objective: In this study, we aimed to investigate the knowledge of healthcare professionals regarding anaphylaxis. Methods: This cross-sectional study, which was conducted between February 2022 and March 2022, included healthcare professionals working in various hospitals in Turkey. A survey consisting of 21 questions which concerned with the demographic data, personal experience and level of knowledge about anaphylaxis was applied to healthcare professionals. Results: The study included a total of 301 participants, comprising 160 specialist physicians (53.16%), 86 resident physicians (28.57%), 31 family physicians (10.3%), and 24 allied health personnel (7.97%). Most of the healthcare professionals (93%) chose epinephrine as the first-line treatment for anaphylaxis. Two hundred ten participants (69.77%) knew the correct dose of epinephrine in the treatment of anaphylaxis, and allied healthcare professionals had least knowledge ( p = 0.009). The participants who received anaphylaxis training and had experience with anaphylaxis had a higher knowledge about epinephrine dosing ( p < 0.001 and p = 0.003, respectively). Only 49.17% of the participants knew the epinephrine doses of EAIs, and only 19% of participants had prescribed an EAI. Conclusion: Our results showed that healthcare professionals' knowledge about epinephrine doses in the treatment of anaphylaxis was not sufficient. Furthermore, the prescribing rate of EAIs was still inadequate. There is a need for national training programs to increase and update the knowledge of healthcare professionals to reduce anaphylaxis mortality.