Gold nanoclusters (AuNCs) are promising nanomaterials for ratiometric fluorescent probes due to their tunable fluorescence wavelengths dependent on size and structure, as well as their biocompatibility and resistance to photobleaching. By incorporating an additional fluorescence spectral peak, dual-emission AuNC-based fluorescent probes have been developed to enhance the signal output reproducibility. These probes can be fabricated by integrating various luminescent nanomaterials with AuNCs. This review focuses on the preparation methods and applications of ratiometric fluorescent probes derived from AuNCs and other fluorescent nanomaterials or fluorescent dyes for both in vitro and in vivo bioimaging of target analytes. Additionally, the review delves into the sensing mechanisms of AuNC-based ratiometric probes, their synthetic strategies, and the challenges encountered when using AuNCs for ratiometric bioimaging. Moreover, we explore the application of protein-stabilized AuNCs and thiolate-capped AuNC-based ratiometric fluorescent probes for biosensing and bioimaging. Two primary methods for assembling AuNCs and fluorophores into ratiometric fluorescent probes are discussed: triggered assembly and self-assembly. Finally, we address the challenges and issues associated with ratiometric bioimaging using AuNCs and propose future directions for further advancing AuNCs as ratiometric imaging agents.