Microbially contaminated food can cause serious health hazards and economic losses, therefore sensitive, rapid, and highly specific visual detection is called for. Traditional detection of microorganisms is complex and time-consuming, which cannot meet current testing demands. The emergence of paper-based biosensors provided an effective method for efficient and visual detection of microorganisms, due to its high speed, all-in-one device, low cost, and convenience. This review focused on 5 biomarkers, namely nucleic acids, proteins, lipopolysaccharides, metabolites, and the whole microorganism of microorganisms. Besides, the recognition methods were summed up in 5 forms, including immunological recognition, aptamer recognition, nucleic acid amplification-mediated recognition, DNAzyme recognition and clustered regularly interspaced short palindromic repeats mediated recognition. In addition, we summarized the applications of paper-based biosensors in the detection of microorganisms thoroughly. Through the exploration of different biomarkers, identification methods, and applications, we hope to provide a reference for the development of paper-based biosensors and their application in safeguarding the food chain.