Nattokinase (NK) is an alkaline serine protease with strong thrombolytic activity produced by Bacillus spp. or Pseudomonas spp. It is a potential therapeutic agent for thrombotic diseases because of its safety, economy, and lack of side effects. Herein, a comprehensive summary and analysis of the reports surrounding NK were presented, and the physical-chemical properties and producers of NK were first described. The process and mechanism of NK synthesis were summarized, but these are vague and not specific enough. Further results may be achieved if detection techniques such as multi-omics are used to explore the process of NK synthesis. The purification of NK has problems such as a complicated operation and low recovery rate, which were found when summarizing the techniques to improve the quality of finished products. If multiple simple and efficient precipitation methods and purification materials are combined to purify NK, it may be possible to solve the current challenges. Additionally, the application potential of NK in biomedicine was reviewed, but functional foods with NK are challenging for acceptance in daily life due to their unpleasant odor. Accordingly, multi-strain combination fermentation or food flavoring agents can improve the odor of fermented foods and increase people's acceptance of them. Finally, the possible future directions focused on NK studies were proposed and provided suggestions for subsequent researchers.