Background Anaphylaxis during anesthesia is a rare but often a potentially life-threatening event for patients. Identifying culprit agents responsible for anaphylaxis is of great important for avoiding potential re-exposure to allergens, but it poses great challenge for anesthetists. This retrospective study aimed to analyze the culprits of patients with a history of perioperative anaphylaxis referred to an anesthesia allergy clinic in China, and to evaluate the role of allergy diagnostic tests in clinical practice. Methods A total of 145 patients (102 female/43 male) who attended the Anesthesia Allergy Clinic for allergen detection between 1 January 2009 and 31 December 2020 were reviewed retrospectively. Clinical characteristics, results of allergy diagnostic tests including skin, and/or basophil activation tests, and the incidence of repeat anaphylaxis after use of recommended alternative anesthetics were obtained. Results Of these 145 patients, 109 patients (75.2%, 74 females/35 males) were determined to experience perioperative anaphylaxis. The most common presenting clinical feature was cardiovascular manifestations (n = 63, 57.8%). According to diagnostic work up, the most common causative agents for perioperative anaphylaxis were neuromuscular blocking agents (n = 35, 32.1%). After diagnostic work up, 52 patients underwent repeat anesthesia. None of these patients experienced recurrent anaphylaxis. Conclusions This study suggests that neuromuscular blocking agents are the main cause of perioperative anaphylaxis. For patients with perioperative anaphylaxis, allergy diagnostic tests are essential to identify causative agents, and to find suitable alternative drugs for the future planning of subsequent anesthetics.