The invention provides a preparation method and application of a V-shaped groove of a micro-fluidic chip injection mold and a preparation method of the micro-fluidic chip, and relates to the field ofmanufacturing of micro-fluidic chips. The preparation method of the V-shaped groove comprises the following steps of providing a third micro conical electrode with a conical tip to machine the workpiece to obtain a main body contour of the V-shaped groove, and then further trimming the third micro conical electrode into a fourth tip conical electrode with a smaller cone height and a smaller angleso as to further modify the body contour of the V-shaped groove to obtain the V-shaped groove with a smaller bottom end round angle. The preparation method solves the technical problem of lack of a high-quality process for manufacturing the V-shaped groove of the micro-fluidic chip injection mold in the prior art.