Gangliosides play vital biological regulatory roles and are associated with neurological system diseases, malignancies, and immune deficiencies. They have received extensive attention in developing targeted drugs and diagnostic markers. However, it is difficult to obtain enough structurally defined gangliosides and analogs especially at an industrial-relevant scale, which prevent exploring structure-activity relationships and identifying drug ingredients. Here, we report a highly modular chemoenzymatic cascade assembly (MOCECA) strategy for customized and large-scale synthesis of ganglioside analogs with various glycan and ceramide epitopes. We typically accessed five gangliosides with therapeutic promising and systematically prepared ten GM1 analogs with diverse ceramides. Through further process amplification, we achieved industrial production of ganglioside GM1 in the form of modular assembly at hectogram scale. Using MOCECA-synthesized GM1 analogs, we found unique ceramide modifications on GM1 could enhance the ability to promote neurite outgrowth. By comparing the structures with synthetic analogs, we further resolved the problem of contradicting descriptions for GM1 components in different pharmaceutical documents by reinterpreting the exact two-component structures of commercialized GM1 drugs. Because of its applicability and stability, the MOCECA strategy can be extended to prepare other glycosphingolipid structures, which may pave the way for developing new glycolipid drugs.