By means of ultraviolet-visible (UV-Vis) and fluorescence spectra, the binding ratio between vitamin K3 and herring-sperm DNA in a physiological pH environment (pH = 7.40) was determined as nK3: nDNA = 2:1, and the binding constants of vitamin K3 binding to DNA at different temperatures were determined as Kθ298K = 1.28 × 105 L.mol−1 and Kθ310K = 7.19 × 104 L.mol−1, which were confirmed using the double reciprocal method are ΔrHmθ = −3.57 × 104 J.mol−1, ΔrGmθ = −2.92 × 104 J.mol−1, and ΔrSmθ = 217.67 J.mol−1K−1. The driving power of this process was enthalpy. An intercalation binding of the vitamin K3 with DNA was supported by a competitive experiment using acridine orange (AO) as a spectral probe. By combination analysis of the Scatchard method and cyclic voltammetry, we suggested that the interaction mode between vitamin K3 and herring-sperm DNA would be a mixed mode. The quinonoid, duality fused-ring of vitamin K3 can intercalate into the base pairs of DNA, and there is an electrostatic binding along with intercalation binding.