The invention discloses a preparation method of an electrochemical aptamer sensor applied to myoglobin detection and relates to the technical field of electrochemical sensors. Polypeptide and HAuCl4 are dissolved in deionized water to obtain solution, and the solution is placed at negative 15 DEG C, is agitated for 12 hours and then reacts with NaBH4 under environments at 0-5 DEG C to obtain a polypeptide -Au nanometer composite material; the polypeptide -Au nanometer composite material is modified on the surface of an electrode and then is covalently connected with myoglobin aptamer to obtain the aptamer sensor applied to myoglobin detection. The detection range of the electrochemical aptamer sensor is wide, the repeatability is good, the influence of common interference substances in meat is avoided, the results are accurate and the electrochemical aptamer sensor can be applied to the detection of myoglobin in meat.