Modified amphoteric polyurethane can be used in the retanning process of leather tanned by organic chrome-free tanning agent because of it’s structure is similar to leather collagen polypeptide and showed good compatibility and reactivity when added into leather. However, most amphoteric polyurethane modifiers have a single function and are derived from non-renewable petrochemical resources, which are not environmentally friendly. Hence, a amphoteric polyurethane was obtained by using isophorone diisocyanate (IPDI) and polytetrahydrofuran diether alcohol (PTMG) as raw materials, 2,2-dimethylolpropionic acid (DMPA) as anionic chain extender, N-methyldiethanolamine (MDEA) as cationic chain extender, then the product (COWPU) is obtained by using castor oil (CO), which is environmentally friendly and has a softening effect on collagen fibers, to modify amphoteric polyurethane. The results showed that COWPU was successfully prepared. It also has significant amphiprotic characteristics (isoelectric point 6.29) and good biodegradability (BOD5/COD=0.37). Then it was applied to retanning of leather tanned by chrome-free tanning agent TWS. The results showed that the tear strength (36.32 N/mm) and tensile strength (9.99 Mpa) of the leather retanned by COWPU were better than the leather retanned by the commercially available amphoteric acrylic retanning agent. In addition, COWPU has a certain softening effect on leather, which has the potential to realize an integrated process of fatliquoring and retanning. This research can provide a matching amphoteric retanning agent for leather tanned by organic chrome-free tanning system.