The invention discloses a temperature-controllable electrochemical DNA (Deoxyribonucleic Acid) sensor based on enzymatic catalysis and a preparation method of the temperature-controllable electrochemical DNA sensor. The temperature-controllable electrochemical DNA sensor is prepared from a golden disk heated electrode, a thiolated single strand DNA probe CP, a target DNA, a signal probe SP labelled with biotin and horseradish peroxidase labelled with streptavidin. The preparation method comprises the following steps: immobilizing a piece of thiolated single strand DNA on the surface of the golden disk heated electrode for capturing the probe CP; complementing one end of the target DNA with the CP; complementing the other end of the target DNA with the a signal probe SP of which the tail end is labelled with the biotin, thus forming a sandwich structure; specifically combining the biotin marked on the SP with the streptavidin coupled with the horseradish peroxidase; immobilizing the horseradish peroxidase on the surface of the electrode; carrying out catalytic reduction on hydrogen peroxide by using the horseradish peroxidase for oxidizing anhaline into benzoquinone; then reducing the benzoquinone into the anhaline on the surface of the electrode. A detecting method for detecting the DNA, provided by the invention, has the characteristics of low cost and high detection sensitivity.