Homologous recombination is a central mechanism for maintaining genome stability and biodiversity. RecA, as the first discovered homologous recombinase, plays a crucial role in homologous recombination strand exchange. In recent years, with the development of structural biology, significant breakthroughs have been made in understanding the static structure of the RecA nucleoprotein filament. However, research on the kinetic process of homologous recombination strand exchange mediated by RecA continues to encounter significant challenges. Research into the dynamic process has been ongoing for decades. In recent years, the use of single-molecule techniques has resulted in significant breakthroughs in this field. Among these techniques, single-molecule fluorescence resonance energy transfer (FRET) technology is widely used due to its ultra-high temporal and spatial resolution, making it well suitable for studying RecA-mediated homologous recombination strand exchange. However, the fluorescent labels required for FRET experiments may affect the RecA-mediated strand exchange process, which is often overlooked by researchers. Most of related articles focus on the effect of fluorescent labels on local structure. This paper primarily examines the effect of DNA fluorescent labeling on protein function, focusing on its effects on strand exchange from two perspectives: strand specificity and conformational sensitivity of the fluorescent labeling. Using experiments such as double-strand binding, single-strand invasion, and strand exchange, we develop a labeling scheme with the minimal effect—9 bp spaced C-strand double-base labeling in triplet— that can effectively improve the efficiency of studying the homologous recombination process. This result enhances the understanding of the effect of fluorescent labeling, allowing researchers to rapidly optimize the position and method of fluorescent labeling, and reduce its negative effects on the strand exchange process. Moreover, it provides some inspirations for other fluorescent labeling experiments.