The fluorescent probe is a powerful tool for biological sensing and optical imaging, which can directly display analytes at the molecular level. It provides not only direct visualization of biological structures and processes, but also the capability of drug delivery systems regarding the target therapy. Conventional fluorescent probes are mainly based on monomer emission which has two distinguishing shortcomings in practice: small Stokes shifts and short lifetimes. Compared with monomer-based emission, excimer-based fluorescent probes have large Stokes shifts and long lifetimes which benefit biological applications. Recent progress in excimer-based fluorescent sensors (organic small molecules only) for biological applications are highlighted in this review, including materials and mechanisms as well as their representative applications. The progress suggests that excimer-based fluorescent probes have advantages and potential for bioanalytical applications.