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Biofilms are the universal lifestyle of bacteria enclosed in extracellular polymeric substances (EPS) on the contact surfaces of food processing facilities. The EPS-encapsulated foodborne bacterial pathogens are the main food contaminant sources, posing a serious threat to human health. The microcrystalline, sophisticated and dynamic biofilms necessitate the development of conventional microscopic imaging and spectral technology. Nanosensors, which can transfer the biochemical information into optical signals, have recently emerged for biofilm optical detection with high sensitivity and high spatial resolution at nanoscale scopes. Therefore, the aim of this review is to clarify the main detection scope in biofilms and the detection principles of optical nanosensors arousing Raman enhancement, fluoresce conversion and color change. The difficulties and challenges of biofilm characterization including the secretion and variation of main biochemical components are first discussed, the details about the principles and application examples of bioassays targeting foodborne pathogens based on optical nanosensors are then summarized. Finally, the challenges and future trends in developing optical nanosensors are also highlighted. The current review indicates that optical nanosensors have taken the challenges of detecting biofilm in complex food samples, including the characterization of biofilm formation mechanism, identification of microbial metabolic activities, diagnosis of potential food pathogens and sanitation monitoring of food processing equipment. Numerous in-depth explorations and various trials have proven that the bioassays based on multifunctional optical nanosensors are promising to ensure and promote food safety and quality. However, there still remains a daunting challenge to structure reproducible, biocompatible and applicable nano-sensors for biofilm characterization, identification, and imaging. [ABSTRACT FROM AUTHOR]