Fluorescence-assisted tools based on organic molecules have been extensively applied to interrogate complex biological processes in a non-invasive manner with good sensitivity, high resolution, and rich contrast. However, the signal-to-noise ratio is an essential factor to be reckoned with during collecting images for high fidelity. In view of this, the wash-free strategy is proven as a promising and important approach to improve the signal-to-noise ratio, thus a thorough introduction is presented in the current review about wash-free fluorescent tools based on organic molecules. Firstly, generalization and summarization of the principles for designing wash-free molecular fluorescent tools (WFTs) are made. Subsequently, to make the thought of molecule design more legible, a wash-free strategy is highlighted in recent studies from four diverse but tightly binding aspects: (1) special chemical structures, (2) molecular interactions, (3) bio-orthogonal reactions, (4) abiotic reactions. Meanwhile, biomedical applications including bioimaging, biodetection, and therapy, are ready to be accompanied by. Finally, the prospects for WFTs are elaborated and discussed. This review is a timely conclusion about wash-free strategy in the fluorescence-guided biomedical applications, which may bring WFTs to the forefront and accelerate their extensive applications in biology and medicine.