The invention relates to a preparation method of an anti-reflection glass substrate. The preparation method comprises the following steps: a, preparing an anti-reflection sol, namely, acidizing silica sol, adding orthosilicate to react with deionized water to form sol, then adding an organic solvent, stirring, sealing and ageing; adding a coalescing agent in the aged sol and controlling a dry chemical additive, and stirring to obtain the anti-reflection sol; b, coating, namely, applying the anti-reflection sol in the step a on the surface of a glass substrate to form a glass substrate with an anti-reflection coating; c, tempering, namely, tempering the glass substrate prepared by the step b to form an anti-reflection film layer; and d, spreading an anti-fouling solution, namely, applying the anti-fouling solution above the film layer of the glass substrate treated in the step c to form an anti-fouling layer. The invention further relates to an anti-reflection glass substrate prepared by adopting the method disclosed by the invention, and an application of the anti-reflection glass substrate.