The invention relates to a construction method of a photo-electrochemical aptamer sensor adopting a TiO2-MoS2-Au ternary composite as a holder. The method comprises steps as follows: firstly, a TiO2 ball template is prepared with a sol-gel method, anatase TiO2 balls with rough surfaces are prepared from the TiO2 ball template, then MoS2 nanosheets are tightly loaded to the surfaces of the TiO2 balls with a hydrothermal method, and a TiO2-MoS2 heterojunction structure is formed; finally, Au nano-particles are deposited on the surface of the heterojunction structure with a trisodium citrate reduction method, and the TiO2-MoS2-Au ternary composite is prepared. The ternary composite has large specific surface area and good biocompatibility, the loading capacity of biomolecules is greatly increased, and the ternary composite further has excellent electrical conductivity and higher photoelectric conversion efficiency, thereby being suitable for constructing a biosensor. DNA aptamers are immobilized on the surface of the TiO2-MoS2-Au ternary composite, and research proves that the photo-electrochemical aptamer sensor prepared with the method can rapidly detect kanamycin and is higher in sensitivity, larger in linear range and lower in limit of detection.