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Microcystin-leucine arginine (MC-LR) is the most toxic and abundant microcystin produced by cyanobacteria. Previous studies have demonstrated that MC-LR can lead to DNA damage by increasing intracellular reactive oxygen species content to induce oxidative stress. However, the direct effect of MC-LR on DNA has not been fully described. In this study, the direct effect of MC-LR on DNA was explored by using spectral analysis and molecular biology technology. First, the fluorescent probe Bptp-R2 was developed to monitor different types of DNA and explore the direct interaction between DNA and MC-LR. The significant differences in the fluorescence of probe–plasmid DNA and probe–ds DNA at various MC-LR concentrations (0, 5, 10, 20, and 30 μmol/L) and MC-LR exposure times (0, 6, 12, and 24 h) showed that the direct interaction between DNA and MC-LR was significant (P ≤ 0.01). Gel electrophoresis demonstrated that the direct interaction between DNA and MC-LR cannot cause DNA strand breaks or change DNA configuration. Then, PCR experiments revealed that the direct interaction between DNA and MC-LR cannot affect DNA replication in a PCR system (P ≤ 0.01). This study discovered that the effects of MC-LR on DNA originate mainly from the secondary effects of MC-LR rather than from the direct interaction between DNA and MC-LR. [Display omitted] • The fluorescence responses of the developed probe to various types of DNA significantly differed (P ≤ 0.01). • The developed fluorescent probe demonstrated significant direct interaction between DNA and MC-LR (P ≤ 0.01). • Gel electrophoresis illustrated that the direct interaction between DNA and MC-LR cannot cause DNA strand breakage or change DNA configuration. • The direct interaction between DNA and MC-LR did not influence DNA replication in the PCR system (P ≤ 0.01). [ABSTRACT FROM AUTHOR]