The growing demand for analysis of the genomes of many species and cancers, for understanding the role of genetic variation among individuals in disease, and with the ultimate goal of deciphering individual human genomes has led to the development of non-Sanger reaction-based technologies towards rapid and inexpensive DNA sequencing. Recent advancements in new DNA sequencing technologies are changing the scientific horizon by dramatically accelerating biological and biomedical research and promising an era of personalized medicine for improved human health. Two single-molecule sequencing technologies based on fluorescence detection are already in a working state. The newly launched and emerging single-molecule DNA sequencing approaches are reviewed here. The current challenges of these technologies and potential methods of overcoming these challenges are critically discussed. Further research and development of single-molecule sequencing will allow researchers to gather nearly error-free genomic data in a timely and inexpensive manner.