摘要： A biomarker-based detection improves early diagnosis and subsequent treatment of diseases. Chemo/biosensing and bioimaging play a crucial role in biomarker detection. Ratiometric fluorescence (FL) probes provide built-in self-calibration for the correction of various target-independent factors and attract particular attention for analytical sensing and optical imaging. Based on the potential to provide a precise, quantitative, visual and real-time analysis, ratiometric FL probes are widely employed for chemo/biosensing and bioimaging detection of biomarkers. In this review, we systematically summarize the recent progress on dual-emission ratiometric FL probes. First, two general categories of ratiometric FL probes are introduced, involving ratiometric FL with one reference signal and two reversible signal changes. Then, fabrication methods of ratiometric FL probes are summarized in detail, including nanoparticle- or organic dye-embedded probes with dual-emission FL, fluorescent nanoparticles and organic dyes dual-embedded probes. Analytical applications of ratiometric FL probes are described overall, referring to different biomarker types and detection mechanisms, ratiometric FL chemo/biosensing in external samples and FL visual detection, ratiometric FL bioimaging in cells and tissues of small animals. Current states, probable challenges and future perspectives of ratiometric FL probes for biomarker detection are discussed rationally. This comprehensive review is attractive for numerous scientists from different areas, such as material science, chemistry, biomedicine, engineering, physics, etc. This review covers a wide range of scientific disciplines and benefits further development of functionalized optical materials, luminescent devices and bioimaging technique.