From June 28 to November 22, 2018, the Chinese People’s Liberation Army Navy – PLA(N) – Peace Ark hospital ship had conducted Mission Harmony 2018, providing humanitarian medical assistance and carrying out international cooperation, in 4 Pacific island countries and 6 Central and South American countries. Compared with its application only in onshore outreach medical teams in the previous Mission Harmony, portable ultrasonography was used both onboard and onshore in Mission Harmony 2018. The purpose of this study was to assess the performance of onboard portable ultrasonography in PLA(N) Peace Ark hospital ship during Mission Harmony-2018, share our onboard working experience, and provide a reference for humanitarian assistance missions in the future. A retrospective review was performed on a cohort of patients checked by onboard portable ultrasonography. Patients’ gender, age, the distribution of examined organs, and multiple applications of the portable ultrasonography were analyzed. Some limitations of portable ultrasonography on the mission and possible improvements in the future were also discussed. A total of 5277 cases (mean age: 43.74 years; range: 2 months–105 years) of ultrasound examinations were performed during the mission; among them, 3126 (59.2%) cases were performed by portable ultrasonography, including 3024 onboard cases and 102 onshore cases. The portable ultrasonography had been applied in many scenarios, for example, onboard emergency triage process, onboard bedside medical support, and onshore outreach medical service, which had become one of the indispensable auxiliary examination methods for its compatibility, portability, and flexibility. The onboard deployment of portable ultrasonography played a versatile and irreplaceable role in the humanitarian medical assistance and medical cooperation carried out by the PLA(N) Peace Ark hospital ship, and will contribute to such kind of missions in the future.