A method of preparing a biological artificial ligament, comprising the following steps: 1) screening of materials: collecting fresh animal ligaments and tendons as a substrate; 2) pretreatment: initially sterilizing the substrate using a broad spectrum, highly-effective, low-toxicity bactericide, and then trimming irregular portions of the substrate; 3) defatting: extracting fatty substances in the substrate with organic solvents using known tissue-treatment techniques; 4) minimizing antigens: treating the substrate with an active reagent to block the specific active groups, such as -OH, -NH 2, -SH, etc., in the proteins of the substrate, and then treating the substrate with a hydrogen bonding reagent with strong hydrogen bonding power to replace the specific hydrogen bonding in the spiral chains of the protein molecules in the substrate and to alter its specific conformation; and 5) fixation: crosslinking and fixing the protein molecules in the substrate using a fixative, 6) coupling of active layer: on the surface of said substrate an active coating is formed by coupling with a coupling agent so as to adhere one or more polypeptides consisting of 16 lysines, glycine, arginine, aspartic acid, serine, proline and cysteine; wherein said active reagents are organic acid anhydrides, acyl chlorides, amides or monocyclic epoxides; the hydrogen bonding reagent forming strong hydrogen bonds comprises guanidine; said fixative is an epoxy compound, a compound comprising two amide groups, a diisocyanate, or a carbodiimide, which easily crosslinks protein molecules; and the coupling agent is a compound comprising two amide groups, or a dioic anhydride compound, or a diepoxy compound, or other compounds with double functional groups which participate in a condensation reaction with groups such as -NH 2, -OH, or -COOH. 1) screening of materials: collecting fresh animal ligaments and tendons as a substrate; 2) pretreatment: initially sterilizing the substrate using a broad spectrum, highly-effective, low-toxicity bactericide, and then trimming irregular portions of the substrate; 3) defatting: extracting fatty substances in the substrate with organic solvents using known tissue-treatment techniques; 4) minimizing antigens: treating the substrate with an active reagent to block the specific active groups, such as -OH, -NH 2, -SH, etc., in the proteins of the substrate, and then treating the substrate with a hydrogen bonding reagent with strong hydrogen bonding power to replace the specific hydrogen bonding in the spiral chains of the protein molecules in the substrate and to alter its specific conformation; and 5) fixation: crosslinking and fixing the protein molecules in the substrate using a fixative, 6) coupling of active layer: on the surface of said substrate an active coating is formed by coupling with a coupling agent so as to adhere one or more polypeptides consisting of 16 lysines, glycine, arginine, aspartic acid, serine, proline and cysteine; wherein said active reagents are organic acid anhydrides, acyl chlorides, amides or monocyclic epoxides; the hydrogen bonding reagent forming strong hydrogen bonds comprises guanidine; said fixative is an epoxy compound, a compound comprising two amide groups, a diisocyanate, or a carbodiimide, which easily crosslinks protein molecules; and the coupling agent is a compound comprising two amide groups, or a dioic anhydride compound, or a diepoxy compound, or other compounds with double functional groups which participate in a condensation reaction with groups such as -NH 2, -OH, or -COOH. on the surface of said substrate an active coating is formed by coupling with a coupling agent so as to adhere one or more polypeptides consisting of 16 lysines, glycine, arginine, aspartic acid, serine, proline and cysteine; wherein said active reagents are organic acid anhydrides, acyl chlorides, amides or monocyclic epoxides; the hydrogen bonding reagent forming strong hydrogen bonds comprises guanidine; said fixative is an epoxy compound, a compound comprising two amide groups, a diisocyanate, or a carbodiimide, which easily crosslinks protein molecules; and the coupling agent is a compound comprising two amide groups, or a dioic anhydride compound, or a diepoxy compound, or other compounds with double functional groups which participate in a condensation reaction with groups such as -NH 2, -OH, or -COOH.