A series of novel hierarchical ZSM-5 zeolite membranes with tunable mesopore sizes have been prepared successfully by using amphiphilic organosilane 3-[(trimethoxysilyl) propyl]octyldimethyl-ammonium chloride (TPOAC) as the mesogenous template and part of the Si source. The effects of several synthesis parameters, such as the TPOAC/SiO2 ratio, synthesis temperature and crystallization time, on the microstructures and rejection performance of the hierarchical ZSM-5 zeolite membranes were systematically investigated. Multiple techniques, such as XRD, SEM, N2 adsorption techniques, TEM, IR, 13C MAS NMR and 29Si MAS NMR were employed for the characterizations. The filtration of dextran in water shows that the membranes have molecular weight cutoff (MWCO) ranging from 9500 to 943,000, corresponding to the molecular size in the range of 4.5–37nm. The sharp increase in the rejection of dextran with the increase of molecular weight indicated narrow pore size distribution of the membrane. These results show that the hierarchical mesoporous ZSM-5 zeolite membranes have a great potential for ultrafiltration with high performance.