The invention discloses a biological colorimetric sensor, a preparation method and application in glucose detection. The preparation method comprises the following steps: putting a mixture of a glucose solution and a glucose oxidase solution into a buffer solution, performing culture, adding a methylene blue solution, uniformly mixing, and further adding an ssDNA (Single-Stranded DeoxyribonucleicAcid) buffer solution, thereby obtaining a mixed solution, namely the biological colorimetric sensor. Compared with the prior art, the biological colorimetric sensor preparation method disclosed by the invention is low in cost and simple to design, the colors of solutions can be changed with glucose of different concentrations, and the change can be read with naked eyes. Under interaction of methylene blue and single-chain DNA, and by virtue of conformation interconversion of the single-chain DNA with polycytosine, a sensor for detecting glucose can be prepared. Results show that the sensor iscapable of sensitively detecting glucose with the concentration of 5mu M to 0.8mM, and also is simple to operate, high in sensitivity and lo win detection limit.