The invention discloses a gene detection chip and a manufacturing method and a detection method thereof. A data line in the gene detection chip has a multilayer structure with a plurality of film layers. The first film layer of the data line is arranged on the same layer as an electrode; the material of the first film layer is a first type metal material; the material of at least one film layer other than the first film layer in the plurality of film layers of the data line is a second type metal material; and the resistivity of the second type metal material is smaller than that of the firsttype metal material. As such, by setting the data line to a multilayer structure, the first film layer of the data line can be made of the same material as the electrode at the same layer, and the metal material with good matching with the gene molecule but high resistivity is adopted, while the other film layers of the data line are made of the metal material with low resistivity, so that the matching of the electrode and the low resistance of the data line can be ensured and the noise of the gene detection can be reduced.