Background: During disasters and emergencies, healthcare systems, particularly hospitals, as the leading providers of care, play an important role; accordingly, this study examines the preparedness of hospitals affiliated with Shahid Beheshti University of Medical Sciences for biological incidents. Materials and Methods: This descriptive cross-sectional study assessed the preparedness level of 14 teaching hospitals in response to biological incidents in 2022. The preparedness of the hospitals in facing biological incidents was evaluated by the secretaries of the hospitals’ risk and disaster management committees using a standard questionnaire (hospital preparedness questionnaire in response to biological incidents) to assess different dimensions of hospital preparedness, including the development of managerial structures, capacity building, training and practice, information and communication management, healthcare system and laboratory, safety and security, patient management, and hospital resilience. Results: The assessment of different hospital preparedness dimensions in facing biological incidents showed that the mean scores of the managerial structure development, capacity building, training and practice, information and communication management, safety and security, healthcare system and laboratory, patient management, and hospital resilience were 20.12, 44.71, 14.14, 19.21, 46.64, 20.42, 27.42, 15.92, respectively. The highest score was related to safety and security and information and communication management, with average scores of 50.78 and 22, respectively. Also, the dimension of training and practice, with an average score of 16.28 and an average percentage of 64.29%, attained the lowest score among the eight dimensions of hospital preparedness. The net score of hospital preparedness in response to biological incidents was obtained at 230.28. According to the final score of hospital preparedness, 9(64.3%) hospitals had a good preparedness status, while 5(35.7%) hospitals acquired an average level of preparedness. Conclusion: The preparedness of the investigated hospitals in response to biological events was favorable. However, coping with biological incidents mandates the highest level of preparedness; therefore, it is necessary to maintain good preparedness and try to improve the condition to an optimal level by focusing on all dimensions, especially those acquiring lower scores.