The present invention discloses a three-dimensional chitosan material preparation method, which comprises: 1) dissolving chitosan in a 10-15% dilute acetic acid solution to prepare a chitosan-dilute acetic acid solution with a concentration of 15-20%, filtering, and carrying out standing defoaming; 2) according to the required shape, uniformly coating the chitosan-dilute acetic acid solution on the inner surface of a corresponding mold, placing the mold into a sodium hydroxide solidification liquid with a concentration of 12-18%, and solidifying at a room temperature to obtain a layer of a white gel used as a chitosan in-situ self-assembly template; and 3) injecting the chitosan-acetic acid solution into the template, carrying out gradient neutralization in a sodium hydroxide solidification liquid with a concentration of 12-18%, carrying out in-situ self-assembly to obtain a longitudinal highly-ordered three-dimensional chitosan material, taking out, soaking with distilled water to achieve a neutral state, and carrying out vacuum drying. According to the present invention, the preparation method is simple, the conditions are mild, various shaped of bone knitting materials such as knit nails, knit rods, splints and the like can be made, and the non-infectious inflammations caused by the material degradation can be avoided.