This work investigates the macromolecular structure of native and amylomaltase (AM) treated cassava starch at various reaction times. AM-treated starches showed lower amylose content compared to their parental starch. Long chain proportions (DP 25–80) increased with reaction time up to 4 h and then slightly decreased at 24 h. Macromolecular and fine structure of AM-treated starches for 5 min (AM5min) and 4 h (AM4 h) were characterized more deeply by using HPSEC-MALLS and H1 NMR combined with β-amylolysis and MALDI-TOF-MS. Molar mass and dispersity of both AM-treated starches were lowered. Nevertheless, AM5min had longer external chains whereas AM4 h exhibited a larger and denser macromolecular core. A cyclo-structure with DP 8 was found in both AM-treated starches; however, AM4 h contained cyclo-structures with various sizes (DP 8–32). Finally, by controlling reaction time and substrate constituents, which are important factors affecting the AM action modes, AM-treated starch with various structural features can be obtained.