Systematic understanding of dynamic biological networks is grounded at the molecular level. Knowledge at this level will discover the functions of molecular components and their interactions with cells or among the molecules themselves. Single-molecule detection has proved to be an indispensable technique for characterizing molecular interactions in various physiological conditions. Among the single-molecule-detection methods, wide-field microscopy-based fluorescence imaging is unique in that dynamic single-molecule events can be vividly observed in a wide observation field. Such a technique has obvious advantages for direct tracking of the pathways and processes of molecular interactions and giving visual information about the molecular mechanisms of many biological events. We discuss experimental schemes and applications of wide-field fluorescence microscopy in characterizing biological molecular interactions and studying virus-cell or particle-cell interactions.