An ultrasensitive electrochemical biosensor for HPV16 oncogene was explored. Hairpin DNA‐1, which can specifically bind with HPV16 oncogene, was fixed on the surface of gold electrode. Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA‐1 to construct Y‐shaped DNA nanostructure, liberating HPV16 oncogene for target recycling. The 3’ terminus of Y‐shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase. Methylene blue was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal. This signal was increased with the concentration of HPV16 oncogene, and the detection limit of HPV16 oncogene was as low as 0.19 fM.