Objective Penicillin allergy skin test is a common procedure in nursing care. Many patients can only do an allergy skin test on fasting due to the disease condition. However, whether or not the fasting state has any effects on the skin testing results remains unclear. Therefore, this study aimed to determine the effects of fasting on the results of penicillin allergy skin tests. Methods This observational cohort study analyzed the clinical data of 200 patients undergoing a penicillin skin test in the emergency center of The Seventh Affiliated Hospital of Sun Yat-sen University from August 1, 2020, to December 31, 2020, of which 100 cases were fasting patients and 100 were non-fasting patients. The information including gender, age, allergy, skin test results, dizziness, and gastrointestinal symptoms were collected and compared. Result Among the 200 patients, five patients (2.5%) tested positive for the penicillin-allergy skin tests. Three patients (3%) tested positive in the fasting group, while two patients (2%) in the non-fasting group tested positive (Relative Risk: 1.5; 95% Confidence Interval: 0.3055 to 7.397; P=0.65). Conclusion These data suggest that fasting may have limited impacts on the results of penicillin allergy skin tests.