Development of real-time and in situ analytical methods for determination of food-borne pathogens and toxins ingested into human body would be a promising research direction in the food safety area. The present review starts with summarization of the up-to-date progress of the nanomaterial-assisted in vitro detection methods for pathogens and toxins, and finally focused on application of animal bioimaging to in vivo study, including prospective strategies for in vivo quantification of target pathogens or toxins and in vivo investigation of their behaviors inside the living body, with the assistance of real-time and non-invasive optical bioimaging. This review provides the advisory direction for food-safety research, from in vitro to in vivo, along with a prospective discussion of the further development roadmap of the food-safety detection techniques, especially the bioimaging-guided methods for investigation and mediation of food contamination effect to human health.