•In vivo fluorescence imaging is important for disease diagnosis and evaluation.•Strategies for developing fluorescent probes using cyanine dyes are summarized.•Cyanine-based probes and materials provide appropriate tools for biomedical visualization. In vivo fluorescence imaging provides vital information required for biomedical research in living animals, and as such plays an essential role in disease assessment. Unfortunately, due to the lack of coherent approaches, in vivo fluorescence visualization is challenging, wasting not only time but the animals used. In recent years, cyanine-based systems have been found to exhibit attractive optical properties with appropriate bio-functionality and therefore have become go-to probes for in vivo research. Herein, we collected the recent breakthroughs using cyanine-based systems associated with use in living mammals, to provide researchers with the required information to develop appropriate strategies able to meet the increasing stringent requirements for practical applications. We concentrate on 6 sites within the cyanine skeleton, including the: meso-position (1), N-indole (2), phenyl-indole (2), cyclohexane (1), which have been used to design symmetric or asymmetric cyanine structures. Substitutions at the meso-position provide fluorescence probes featuring a switch of EWG and EDG, PET, spiro-cyclization, ICT within the cyanine skeleton, or FRET, for visualizing analytes in vivo. Substitution at the N-indole forms a positively charged system with long wavelength emission suitable for in vivo imaging. While, modification of the phenyl-indole contributes to the functional properties, including photoacoustic, photothermal and photodynamic behavior, which can be used for multimodal imaging as well as functional imaging in vivo. With a specific trigger at the N-indole or meso-position, NIR fluorescence pro-drugs could be developed for in vivo tracing of the efficiency of drug delivery. While modification of the cyclohexane can introduce a targeting moiety for the accurate monitoring of tumors or organs. Using these strategies appropriate design platforms can be developed to provide the next generation of optical systems for living animal research. Moreover, we hope this review will enable researchers to design systems with high efficiency for in vivo research, which will reduce both the development time and number of animals required to progress the research.