The precision of analytical methods using fluorescent probes or biomolecular labels often is compromised of a variety of conceivable interferences that may originate from the instrumental system, the sample, or the underlying sensor chemistry. Instrumental drifts of the optoelectronic system, photobleaching of luminophores, or high intrinsic color and background fluorescence of the sample cannot be eliminated even by extensive calibration procedures. As a result, intrinsically referenced methods are preferred to improve optical chemo- or biosensor technology, and fluorescent bioimaging. Intrinsic referencing is often accomplished by ratiometric techniques. These include (1) dual wavelength probes, (2) dual luminophore sensors, (3) lifetime-based assays, (4) dual lifetime referencing (in the time and frequency-domain), and (5) fluorescence anisotropy. Applications and advantages of the various approaches are outlined in this review, with a focus on widely used sensing methods for oxygen, pH, carbon dioxide, calcium, glucose, or temperature, and on biomolecular screening. In addition to ratiometric methods, fluorescence correlation spectroscopy represents another attractive tool to determine analyte concentrations via fluorescent probes. Many of these ratiometric approaches have the potential to pave the way for the development of calibration-free sensor and imaging schemes.