Starch nanoparticles (SNPs) are innovative food hydrocolloids with improved functionalities for enhancing food texture and nutrition. This study investigates the sequential treatment of pullulanase and ultrasound to produce SNPs from cassava and faba bean starch. First, an optimisation of pullulanase enzyme modification was performed using Response Surface Methodology (RSM) with a Box-Behnken Design (BBD). Then, three processing sequences were compared, including (1) enzyme only, (2) pre-treatment ultrasound followed by enzyme, and (3) enzyme followed by post-treatment ultrasound. RSM-BBD identified optimal conditions for processing 3% starch with 75 U/g enzyme and a 10 h incubation time, which successfully produced SNPs. Sequential enzymatic treatment followed by post-treatment ultrasound yielded the smallest and uniform nanoparticles (cassava: PSD = 134 nm, PDI = 0.220; and faba bean: PSD = 208.7 nm, PDI = 0.451). Size exclusion chromatography results demonstrated a significant molecular weight reduction from the native form (cassava: 1.2×10 ⁷ g/mol, and faba bean: 4.8×10 ⁷ g/mol) to the enzymatic (cassava: 4.7×10 ³ g/mol, and faba bean: 6.8×10 ³ g/mol), with the most reduction observed in the sequential enzymatic and post-treatment ultrasound (cassava: 4.1×10 ³ g/mol, and faba bean: 5.1×10 ³ g/mol). Structurally, the sequential enzymatic ultrasound treatment allowed structural re-organisation. From X-ray diffraction, the formation of a V-type crystalline pattern was observed, followed by a significant increase in relative crystallinity (cassava: 37.9% to 59.6%, and faba bean: 41.5% to 53.1%). Fourier-transform infrared and nuclear magnetic resonance analyses confirmed an increase in short-range structural order, indicating reorganisation toward a more ordered starch structure. Functionally, all enzymatically treated samples developed unique semisolid-forming capabilities at room temperature and a low starch concentration of 3%, representing a novel cold-set gelation mechanism distinct from conventional heat-induced starch gelation. Ongoing rheological characterisation will provide more comprehensive insights into the rheological properties, while digestibility profiles indicated an increase in resistant starch content, supporting applications in semi-solid food systems for better glycemic level management. This research provides a clear mechanistic insight into how enzymatic and ultrasound methods can be utilised to enable the fabrication of innovative food hydrocolloids with improved nutritional and functional properties.