Identified and analyzed highly cited publications in the Web of Science category of biomedical engineering in the last three decades were investigated. Documents that have been cited more than 100 times from Web of Science Core Collection since publication year to the end of 2020 were defined as highly cited publications. The analyzed aspects covered document types, distribution of annual production and its citations per publication, journals, countries, institutes, authors, and the top cited articles. Publication performance of countries and institutions were evaluated by six publication indicators. Y-index was applied to evaluate authors’ publication potential and their publication characteristics. Citation indicators including total citation and citations in 2020 were used to compare the most frequently cited articles. Results shows that most highly cited papers were published in journals with high impact factors. The USA dominated the six publication indicators. The G7 were ranked in the top ten productive countries. Eight of the top ten productive institutes were all located in USA. Results from Y-index shows that highly cited authors had higher proportion of corresponding-author articles and first-author articles. The article by Kokubo and Takadama in 2006 was the most frequently cited and the article by Litjens et al. in 2017 was the most impactful in 2020.