The interaction of a fluorescent nucleotide analogue, 2'-(or-3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), with the Klenow fragment of DNA polymerase I (Pol I) was visualized at a single-molecule level. Upon excitation, individual enzyme-TNP-ATP complexes resulted in bright fluorescent spots owing to the increase of the fluorescence quantum yield of TNP-ATP when it bound to the enzyme molecule, whereas unbound TNP-ATP molecules were not visible in the single molecule detection, Thus, we directly investigated the individual interactions of TNP-ATP with the enzyme using single-molecule fluorescence imaging and time-resolved spectroscopy of single enzyme-TNP-ATP complexes without prior separation of the unbound probe molecules.