The invention provides a preparation method of nano-chitosan for preparing an antibacterial natural rubber composite material. According to the invention, sodium tripolyphosphate is used as an ionic inducer, and vanillin is used as a chemical cross-linking agent. By ion induction combined chemical crosslinking method, chitosan nanoparticles obtained after physical crosslinking of sodium tripolyphosphate are added into vanillin to carry out chemical crosslinking; and cross-linked particles are directly washed with water to neutral so as to obtain a nano-chitosan suspension with particle size range being 160-1000 nm, with average particle size being 250-500 nm and having stable quality. The preparation is performed in a water phase, the reaction condition is wild, and any organic solvent or high-temperature high-pressure radiative complex equipment are not used. The prepared nano-chitosan suspension can be directly added into natural latex so as to obtain natural rubber composite material with an excellent antibacterial property.