The invention relates to method for preparing a functionalized composite nano-fiber modified electrode. The method specifically comprises the steps of (1) preparing a spinning solution; (2) preparing composite nano-fibers through electrostatic spinning so as to form a composite nano-fiber PA6-MWNTs modified electrode; (3) carrying out electrical-polymerization thionine functionalization on the composite nano fibers: soaking the composite nano-fiber PA6-MWNTs modified electrode into a polymerization solution containing thionine monomers PTH, applying a voltage to the modified electrode so as to carry out anodizing treatment, then, carrying out cyclic volt-ampere scanning, washing away the thionine monomers, adsorbed to the modified electrode, by using a phosphate buffer solution PBS, forming a functionalized composite nano-fiber electrode modification material PA6-MWNTs/PTH layer on the surface of the electrode after the polymerization reaction is ended, thus obtaining the functionalized composite nano-fiber PA6-MWNTs/PTH modified electrode. According to the method, a functionalized composite nano-fiber electrode modification material which has the characteristics of good stability, large specific surface area, good biocompatibility, high electron transfer rate, uniformly-distributed diameter and pore size, and the like is obtained.