Optical imaging and photoacoustic (PA) imaging have become essential tools to investigate physiological or pathological processes at the molecular level in vivo. The detection of variations at the molecular level in vivo is particularly important owing to the rapid progression of diseases. However, most studies have mainly focused on plain qualitative molecular imaging and detection, which is characterized by the absence of a reference signal in one-channel responsive imaging. To overcome the limitation and quantitatively detect molecules in situ, this Account reviews the recent contributions of our group to the quantitative imaging field in the form of ratiometric optical and PA imaging in vivo in the second near-infrared window (NIR-II, 950-1700 nm).In this Account, we present recent advances that our group has made in ratiometric imaging probe design and biomedical applications by constructing probes based on ratiometric optical imaging and ratiometric PA imaging. First, we highlight the design strategies of ratiometric optical probes that were based on organic ratiometric molecular probes, radio-activated organic ratiometric probes, and hybrid organic-inorganic assembled ratiometric probes. Subsequently, the design strategies of the ratiometric NIR-II optical nanoprobes with activated bioluminescence resonance energy transfer (BRET), Förster resonance energy transfer (FRET), and nonradiative energy transfer (NRET) effects provide a reliable tool to achieve the ratiometric detection of endogenous signaling molecules and thereby apply it to the monitoring and evaluation of the efficacy of photodynamic therapy, radiotherapy, and immunotherapy to guide the treatment process. In addition, we systematically introduce the functional design principles of ratiometric PA imaging probes based on core-shell nanoprobes, core-satellite nanoprobes, and universal hybrid nanoprobes, where we have established that reference signal and sensing signal can be obtained from the random assortment of plasmonic components and organic semiconducting molecules using a phase separation strategy. On these insights, we discuss the rational and detailed biomedical applications of ratiometric PA imaging probes which include accurate quantitative detection of disease-related molecules in inflammation or tumors in real time. In these champion implementations of ratiometric PA imaging probes, different diagnostic modules have been linked through compound modification with activation characteristics (e.g., pH, redox, enzyme, hypoxia). Finally, we present the challenges and perspectives for ratiometric probes based on optical imaging and PA imaging for multitarget design and future clinical translation. We believe that the upcoming generations of ratiometric imaging probes would have promising potential applications in the precise diagnosis of diseases. Finally, this Account may stimulate innovative studies in the design of ratiometric imaging probes and exploration of their clinical applications.