The invention discloses nanometer blue titanium dioxide colloid and a preparation method and application thereof. The colloid is a nanometer blue titanium dioxide ball, wherein the ball diameter is 20-40nm and the ball is composed of tetravalent titanium ions and trivalent titanium ions according to an atomic percent ratio of 51%: 49%. The preparation method comprises the following steps: firstly, mixing white rutile phase titanium dioxide powder with deionized water according to a weight ratio of (1-10): 1, uniformly stirring to obtain emulsion, and then, irradiating the emulsion by using laser with a wavelength of ultraviolet, power of 0.5-4.5W, frequency of 8-12Hz and a pulse width of 4-8ns for 10-30min to prepare a target product. An aqueous solution of the target product can be used as a detection reagent to detect glucose through a colorimetric method, or a mixed solution of the aqueous solution of the target product, glucose oxidase and a phosphate buffer solution is used as the detection reagent to detect glucose through the colorimetric method; and when used for detection, the target product has the advantages of simple process, good quickness and sensitivity, low detection cost, short time, environmental protection and the detection accuracy is hardly influenced by external factors.