Binding modes of helicase to various DNA substrates were visualized by single-molecule fluorescence imaging on silicate coverslips coated with poly(ethylene glycol) (PEG) which minimizes the background noise from nonspecific protein adsorption. The results clearly show that the helicase has higher affinity for double-stranded DNA (dsDNA) with a single-stranded DNA (ssDNA) tail than that without one and that multiple helicases can bind to 4.7-kilonucleotide (knt) ssDNA. The study reported here will enhance the capabilities of single-molecule fluorescence studies on DNA-protein interactions.