The invention belongs to the technical field of medical instruments, and discloses a glucose sensor electrode as well as a preparation method and application thereof. The method comprises the following steps: (1) performing alkali heat treatment and forming a nano-needle structure titanium dioxide on the surface of a titanium electrode; (2) forming a micro-area groove on the nano-needle structuretitanium dioxide film layer to obtain nano-needle structure titanium dioxide with micropattern; (3) depositing chlorine doped polypyrrole in the groove according to a chronopotentiometry method; (4) forming a nano-cone structure citric acid doped polypyrrole on the chlorine doped polypyrrole by a chronopotentiometry method; (5) grafting enzyme capable of detecting glucose to obtain the glucose sensor electrode. The glucose sensor electrode provided by the invention is used for detecting the glucose rapidly and highly sensitively. The glucose sensor electrode is simple and stable in process andlow in cost, can graft the enzyme capable of detecting the glucose on the surface of the electrode efficiently, has high reliability on glucose detection and has high stability.