The modulation of numerous signaling pathways is orchestrated by redox regulation of cellular environments. Maintaining dynamic redox homeostasis is of utmost importance for human health, given the common occurrence of altered redox status in various pathological conditions. The cardinal component of the thioredoxin system, mammalian thioredoxin reductase (TrxR) plays a vital role in supporting various physiological functions; however, its malfunction, disrupting redox balance, is intimately associated with the pathogenesis of multiple diseases. Accordingly, the dynamic monitoring of TrxR of live organisms represents a powerful direction to facilitate the comprehensive understanding and exploration of the profound significance of redox biology in cellular processes. A number of classic assays have been developed for the determination of TrxR activity in biological samples, yet their application is constrained when exploring the real-time dynamics of TrxR activity in live organisms. Fluorescent probes offer several advantages for in situ imaging and the quantification of biological targets, such as non-destructiveness, real-time analysis, and high spatiotemporal resolution. These benefits facilitate the transition from a poise to a flux understanding of cellular targets, further advancing scientific studies in related fields. This review aims to introduce the progress in the development and application of TrxR fluorescent probes in the past years, and it mainly focuses on analyzing their reaction mechanisms, construction strategies, and potential drawbacks. Finally, this study discusses the critical challenges and issues encountered during the development of selective TrxR probes and proposes future directions for their advancement. We anticipate the comprehensive analysis of the present TrxR probes will offer some glitters of enlightenment, and we also expect that this review may shed light on the design and development of novel TrxR probes.