OBJECTIVE： To prepare nattokinase （NK）-loaded trimethyl chitosan （TMC） nanoparticles （TMC-NK-NPs）， and to study its release rate in vitro. METHODS： TMC with different degree of quaternization （15％， 20％， 25％） were synthetized， i.e. TMC15， TMC20， TMC25； those were combined with NK to form TMC-NK-NPs by self-assembly. The particle size， polydispersity index （PDI） and Zeta potential were determined and TCM was screened； the morphology， entrapment efficiency （EE）， drug-loading amount， 30 d stability at 4 ℃ and dark place， 24 h accumulative release rate in vitro （Q） and NK activity of TMC-NK-NPs by optimal formulation were observed and determined. RESULTS： Using TMC20， TMC-NK-NPs were smaller in particle size [（161.0±4.8） nm]， the lowest in PDI （0.204）； the Zate potential was （19.2±1.5） mV， spherical or spherical-like， EE was （45.4±1.51）％， drug-loading amount was （14.2±0.25）％， stable within 30 d， the peak Q12 h was 92.3％. The maximum released activity of NK was 76.6％ at 1 h， but then gradually decreased with time. CONCLUSIONS： TMC-NK-NPs are prepared successfully， round and complete with high EE and drug-loading amount， good sustained-release property.