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As studies on life sciences progress toward the single-molecule level, new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomole-cules. Optical tweezers and fluorescence microscopy have been combined to solve this problem. The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis. Powerful manipulation and fluorescence visualization have been widely used, and these techniques provide new possibilities for studying complex biochemical reactions at the single-molecule level. This paper explains the features of this combined technique, including the application character-istics of single-trap and dual-traps, the anti-bleaching method, and optical tweezers combined with epi-fluorescence, confocal fluorescence, total intal reflection fluorescence, and other fluorescence methods. Using typical experiments, we analyze technical solutions and explain the factors and principles that instrument designers should consider. This review aims to give an introduction to this novel fusion technology process and describe important biological results.