In order to investigate the effects of microfluidization in different pressure combined with pullulanase debranching treatment on the structural properties and starch composition of lotus seed starch-glycerin monostearin complex, the determination of amylose, complex index, crystal structure, microstructural morphology and starch composition were discussed and analyzed. The results showed that the appropriate microfluidization conditions were more conducive to exert the debranching of pullulanase and facilitate the formation of lotus seed starch-glycerin monostearin complex. 80 MPa of microfluidization condition contributed to the crystal structure of the starch complex developing in the direction of high-microcrystalline V_6I-type, and the formation of the complexes would inhibit the gelatinization of starch caused by the thermal effect, while excessive microfluidization pressure would lead to the incomplete crystallization of starch and the phenomenon of re-gelatinization. On the other hand, the resistant starch proportion of V6I-type complex formed by microfluidization conditions of 80 MPa was significantly higher than that of other sample groups, which is of great significance for the development of new resistant starch.