With the increasing emphasis on ecological safety and physical health, the detection and treatment of harmful substances and diseases are becoming more and more prevalent. Therefore, efficiently monitoring these biological behaviors with high accuracy and sensitivity in real-time has shown prominent research significance. The use of fluorescent probes to analyze organisms has gained momentum in recent years, especially in the field of organ imaging and assisted cancer therapy, where fluorescent bioanalysis demonstrates significant advantageous. In this review, we explored the latest advancements in fluorescent molecular probes (e.g., small-molecule, macro-molecule, supramolecule) and fluorescent nanoparticle probes (e.g., quantum dots or nanoclusters, metal-organic frameworks, polymers, complexes) used as bioanalytical tools in various assays over the last three years. We also delved into their detective mechanisms, specific application areas, and characterization tools for responsive behavior. This review aims to showcase the most recent and comprehensive research progress in fluorescent bioanalysis based on molecular and nanoparticle probes, offering guidance for future developments in the design and fabrication of fluorescent probes and their potential applications.