For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.