Abstract: In view of the problems such as compact surface, small specific surface area and lack of much pore structure of traditional cross-linked lipase aggregates (CLEAs), which further affects the catalytic efficiency of the enzyme in the catalytic process. To solve above problems, this study prepared porous-cross linked enzyme aggregates (p-CLEAs) were synthesized by in-situ co-precipitation method using CaCO3 microparticles as templates. The preparation procedure involves crude lipase was immobilized as CLEAs via precipitation with ammonium sulfate and entrapping this lipase molecules into CaCO3 templates, followed by DTT (dithiothreitol) induced assemble of lipase molecules to from lipase microparticles (Lipase molecules were assembled into a microparticle by the internal using disulfide bonds within lipase molecules as molecular linkers and stimulated by dithiothreitol) and finally the removal of CaCO3 templates by EDTA to form pores in the CLEAs. The preparation conditions, structural characteristics and enzymatic properties were studied. The results showed that the best preparation conditions were: mass concentration of concentration of Ca2+ 0.35 mol/L, the ratio of lipase to Ca2+ is 5:1, saturation of precipitator is 80%, precipitation pH value is 8, precipitation time is 40 min, DTT volume fraction is 0.2%, and crosslinking time is 40 min, Compared with conventional CLEAs, the prepared p-CLEAs presented a porous structure and showed significant improvement in methanol tolerance, thermal stability and pH value stability. It was preserved at 4 ℃ for 6 months and still maintained a high activity. This porous structure makes it easier for substrate molecules to enter the active site of lipase, which not only reduces the mass transfer limitation, but also improves the catalytic efficiency. Therefore, p-CLEAs lipase has a high catalytic activity.