Abstract: To protect nattokinase from inactivation and improve its absorption in gastrointestinal tract,the optimum preparation condition of nattokinase microcapsules formed by double emulsion evaporation method was obtained by orthogonal experiment,with poly(ethylene glycol-b-(DL-lactic acid-co-glycolic acid)-b-ethylene glycol)(PEG-PLGA)as the wall material,and entrapment efficiency as the index. Then nattokinase microcapsules were respectively prepared with PEG-PLGA and folate-poly(ethylene glycol-b-(DL-lacticacid-co-glycolic acid)-b-ethylene glycol)(FA-PEG-PLGA)as the wall material,under the optimum condition. Finally,the gastrointestinal release in vitro and cytotoxicity and absorption in Caco-2 monolayer model of the two microcapsules were evaluated. Results showed that,the optimum preparation condition of PEG-PLGA nattokinase microcapsules were listed as follows:Wall material concentration of 5 mg/mL,PVA concentration of 1%,homogenization speed of 17500 r/min and homogenization time of 7.5 min for double emulsion. For PEG-PLGA and FA-PEG-PLGA nattokinase microcapsules,their average particle sizes were 271.33 and 255.75 nm,the encapsulation efficiency was 61.54%±2.36% and 58.76%±2.54%,the Zeta potential was(-20.17±1.42)and(-24.73±2.36) mV,respectively. Results of gastrointestinal release in vitro showed that more than 60% nattokinase was retained in the two microcapsules after 2 h in gastric pH,and the sustained release effect was good in the intestinal environment(pH7.0)within 22 h. Both microcapsules had almost no toxicity to Caco-2 cells at a concentration of 500 μg/mL,and could be well absorbed,the apparent permeation coefficients of the two microcapsules were as high as 2.367×10^-6 and 3.497×10^-6 cm/s,respectively. Results of laser confocal microscopy also showed that both microcapsules were well absorbed in Caco-2 cells,and FA-PEG-PLGA microcapsules were absorbed better than PEG-PLGA microcapsules.