We present a new protocol for DNA assay under ambient, routine operation. The novelty of the proposed approach is the direct analysis, meaning there is no need for derivatization or other pretreatments which are usually coupled with conventional bio or biochemical assay. The proposed method is based on our new preparation of a nanocomposite thin film composed of nickel hydroxides. Fabrication of the sensing film is facile, in situ, and thickness-controllable, and combines the metal deposition and the subsequent stripping/formation. The film construction and the novel sensing events were thorough examined and characterized by various techniques. Although the responding sensitivities vary among nucleic acids, the sensing mechanism was found to be attributed to the Ni(III/II) moieties within the film. In alkaline media the electrogenerated higher valent Ni(III) would oxidize DNA at around the redox potential of Ni(III/II). This electrocatalytic oxidation thus accounts for the much enhanced voltammetric current and for the much reduced overpotentials of these analytes at unmodified electrodes. The advantage of this protocol stemmed from the utilization of the same medium for both sensor fabrication and subsequent exploration that preserve and maximize the catalytic capacity. In addition to chromosomal DNAs, denatured DNA, single stranded DNA, and synthetic oligomers can all be determined by the proposed protocol with negligible interference. The present work offers a fast, ergonomic, and cost-effective alternative to the existing DNA assay arena.