Fluorescence spectroscopy has emerged as a powerful analytical technique in the food industry for quality and control, offering rapid, affordable user-friendly and non-invasive analysis with high sensitivity and potential for online usage. This chapter explores the utilization of fluorescence spectroscopy techniques, for assessing various aspects of food quality and safety in the Agri-food value chain. The intrinsic fluorescence of food components, including proteins, lipids, and pigments, serves as a valuable indicator for monitoring changes related to freshness, spoilage, and contamination. The application of fluorescence spectroscopy in characterizing the structural and compositional features of food matrices provides insights into the molecular dynamics and interactions that influence product quality. Furthermore, fluorescence spectroscopy has proven as effective tool in detecting and quantifying specific contaminants, such as pesticides, mycotoxins, and bacterial pathogens, contributing to enhanced food safety measures. The technique’s ability to discriminate between different fluorophores enables the identification of adulteration and the evaluation of processing-induced changes, ensuring adherence to regulatory standards. In addition to its analytical capabilities, fluorescence spectroscopy offers real-time monitoring during food processing and storage, enabling proactive intervention to prevent quality deterioration. Recent technological advancements, including the integration of fluorescence imaging and chemometrics, further enhance the precision and speed of analysis, making it applicable across diverse food matrices. This comprehensive write-up highlights the current state of fluorescence spectroscopy in the field of food science and underscores its potential as a reliable, rapid and non-destructive tool for quality assessment and control. As the demand for high-quality and safe food products continues to grow steadily, fluorescence spectroscopy stands ahead as pivotal role in advancing the food industry’s capabilities for quality assurance and consumer protection as well as check food fraud and traceability. The use of fluorescence spectroscopy is due to the application of multivariate statistical analyses that could be classified to descriptive and predictive methods. Some examples of the application of these techniques will be elucidated in the present chapter.