Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption, and visualization capability. Recently, a variety of nucleic acid amplification approaches have been developed to provide a straightforward and highly efficient way for amplifying low abundance target signals. The integration of single-molecule fluorescence imaging with nucleic acid amplification has greatly facilitated the construction of various fluorescent biosensors for in vitro and in vivo detection of DNAs, RNAs, enzymes, and live cells with high sensitivity and good selectivity. Herein, we review the advances in the development of fluorescent biosensors by integrating single-molecule fluorescence imaging with nucleic acid amplification based on enzyme (e.g., DNA polymerase, RNA polymerase, exonuclease, and endonuclease)-assisted and enzyme-free (e.g., catalytic hairpin assembly, entropy-driven DNA amplification, ligation chain reaction, and hybridization chain reaction) strategies, and summarize the principles, features, and in vitro and in vivo applications of the emerging biosensors. Moreover, we discuss the remaining challenges and future directions in this area. This review may inspire the development of new signal-amplified single-molecule biosensors and promote their practical applications in fundamental and clinical research.