Standardized specimen referral with proper packaging and transportation management is crucial in ensuring biosafety and biosecurity. USAID’s Infectious Disease Detection and Surveillance (IDDS) project supported the development of an integrated and multisectoral specimen referral system (SRS) in Vietnam that applies the One Health approach. Informed by assessments of current specimen referral activities across the human and animal health sectors, the development of an SRS in Vietnam was divided into three phases: design, pilot, and evaluation. The pilot was implemented between June 2022 and December 2023 in the three provinces of Thai Nguyen (North), Binh Dinh (Central), and Dong Thap (South), Vietnam. Critical aspects of the SRS under evaluation included specimen delivery time and compliance with biosafety and biosecurity standards in packaging and transportation. The SRS was established with customized services for transporting potentially infectious specimens for 78 human and animal health facilities in three provinces. The customized service for specimen transport provided by a courier company was the predominant mode of specimen transport in the established system. Proper packaging materials and 300 incident handling kits were provided. Eight standard operating procedures and one national guideline were established, and 28 training courses were organized for human and animal health facility staff and the courier company in three provinces and relevant hub locations to ensure proper specimen packaging and transport practices. During the pilot implementation, over 90% of 13,934 specimens were transported within the targeted lead time. The developed SRS system addressed gaps and provided effective integrated and multisectoral specimen referral services for various types of disease specimens in Vietnam to strengthen the diagnosis and surveillance of human and animal infectious diseases.