The invention relates to a preparation method of a multi-layer absorbable biological membrane. The method comprises the steps of preparing a chondroitin sulfate acetic acid solution and a collagen acetic acid swelling solution; adding the chondroitin sulfate acetic acid solution to the collagen acetic acid swelling solution to obtain collagen-chondroitin sulfate slurry; carrying out first vacuum freeze-drying on a collagen chondroitin sulfate solution to obtain a collagen membrane; pressing the collagen membrane into a compact collagen membrane; pouring the collagen chondroitin sulfate solution into a freeze-drying plate paved with the compact collagen membrane or directly adhering a compact collagen layer to the collagen membrane through the collagen-chondroitin sulfate slurry, carrying out second vacuum freeze-drying to obtain a compound collagen membrane; crosslinking the compound collagen membrane; and sterilizing the crosslinked compound collagen membrane to obtain the multi-layer absorbable biological membrane. The multi-layer absorbable biological membrane formed by a loose layer and the compact layer is prepared from high-purity I-type collagen as a raw material; and the multi-layer absorbable biological membrane has good biocompatibility, and is nontoxic and free of antigen reaction.