Abstract: In order to comprehensively summarize the current research status of biological nitrogen fixation (BNF) globally and domestically. The relevant literatures on BNF were retrieved from the Web of Science (WOS) and the China National Knowledge Network (CNKI) databases from 1990 to 2023. By using Citespace, VOSviewer, HistCite, and other bibliometric tools, we analyzed the number of publications, leading countries/regions and institutions, significant journals and researchers, highly cited papers, emerging research hotpots and the challenges faced in the field. The results showed as follows: 1) Over the past 24 years, the number of BNF-related publications has increased annually. Since 2010, Chinese authors have significantly contributed to this field, ranking second in the WOS database. Notably, China has shown strong collaboration with countries like the United States, Brazil, and Germany. Most English-language BNF literature falls within plant science and microbiology, whereas Chinese-language literature is primarily categorized under biology and crop science. The significant journals publishing BNF research include Biotechnology Bulletin (Chinese), Plant and Soil, Soil Biology & Biochemistry. Prominent researchers in this field include Hungria M, Urquiaga S, Boddey R M, and Li Youguo. 2) The keyword clustering network divides BNF research into four key areas, microorganisms and their colonization, the impact of BNF on agricultural production, the molecular mechanism of BNF, and factors influencing BNF. All these research areas focus on enhancing BNF efficiency and broadening its application scope. 3) Future research on BNF faces several challenges, including expanding the host range of rhizobia, improving rhizosphere-associated nitrogen fixation efficiency, introducing BNF into cereal crops to develop “N-self-fertilizing” varieties, exploring efficient nitrogen-fixing microbial resources, and optimizing microbial fertilizer application technology. 4) Technological advancements such as genetic engineering, synthetic biology, metagenomics, and transcriptomics have sparked interdisciplinary research, providing tools for deeper exploration of BNF mechanisms and efficient application for green and low-carbon agricultural production.