Streptavidin–fluorescent proteins (SA-FPs) are a versatile tool to visualize a broad range of biochemical applications on a fluorescence microscope. Although the avidin–biotin interaction is widely used, the use of SA-FPs has not been applied to single-molecule DNA visualization. Here, we constructed 12 bright SA-FPs for DNA staining or labeling reagents. To date, 810 FPs are available, many of which are brighter than organic dyes. In this study, 12 bright FPs were selected to construct SA-FP plasmids covering green to red colors. Their brightness ranges from 40 to 165 mM–1 cm–1. Moreover, SA-FP is brighter than FP itself because streptavidin forms a tetramer complex; thus, four FPs are in a single complex. In addition, FPs often form a dimer or a tetramer, resulting in multiple FPs in a single spot on a microscopic image. This feature is advantageous because multiple fluorescent β-barrels on a single biotin tag provide enough brightness to be easily visualized by epifluorescence microscopy. Using SA-FPs, we visualized DNA backbones, nickase-based optical mapping, and AT-frequency profiling. Finally, we demonstrated the combination of nickase-based optical mapping using SA-FP and AT-frequency profiling.