A detection substrate and a manufacturing method thereof, and a nucleic acid detecting method are provided. The detection substrate includes at least one detection unit disposed on a base substrate, the detection unit including a first electrode, a second electrode and a colloid layer, the second electrode disposed on a side of the first electrode away from the base substrate, the second electrode including at least one hollowed structure, the second electrode and the first electrode being insulated from each other, and the first electrode and the second electrode being configured to be applied with voltages respectively; the colloid layer at least disposed on the at least one hollowed structure of the second electrode, the colloid layer including a linker configured to be paired with a target nucleic acid.