The invention discloses a liver cancer diagnostic kit based on nucleic acid aptamers, comprises the following ingredients with independent packing: liver cancer nucleic acid aptamer solution, polyacrylamide gel mother liquor, ammonium persulfate, tetramethyl diethylamine, 50% glycerin solution II, 1.25M sodium chloride solution, GelRed dye solution, positive control serum, and negative control serum. The kit has the following characteristics: (1) high diagnostic value, sensitivity of about 85%, specificity of about 85%, and accuracy of about 85%; (2) simpleness and practicality, and only need of conventional vertical electrophoresis and a gel image analysis system to realize the diagnosis; and (3) low cost and high additional value. Liver cancer is one of common malignant tumors in clinic, thus by applying the kit of the invention in clinical application, the consumption is high, the diagnostic level of liver cancer can be effectively raised, good economic benefit and social benefit can be produced, and the application prospect is good.