The present disclosure discloses a novel biochip substrate, a preparation method and an application thereof. The surface of the novel biochip substrate contains active vinyl sulfone groups. The preparation method involves a one-step reaction of a compound containing vinyl sulfone groups on both ends with a silicon-hydroxyl group on the surface of a silicon-based biochip substrate material under catalytic conditions, to prepare the biochip substrate. The application immobilizes biomacromolecules by conducting Michael addition of amino or sulfydryl group in biomacromolecules and the vinyl sulfone group on the surface of the biochip substrate, realizing biological functionalization thereof. The biochip substrate has high-density active vinyl sulfone groups, which can be used for immobilization of various biomolecules with mild fixation conditions and simple operation. The preparation method does not require complex pretreatment processes, and has high operability and reproducibility, low cost, mild reaction conditions, simple operation, and environmentally friendly, which is a broad-spectrum biochip substrate with great potential.