The effect of ultrasonic treatment on the structure, size and aggregated morphology of Lignosus rhinocerotis polysaccharide (LRP) was investigated. Ultrasonic treatment for 10 min has demonstrated to improve the aqueous solubility of LRP, leading to a uniform and narrow LRP particle size distribution. Meanwhile, short-time ultrasound was found to obviously decrease the molecular size parameters (Mw, Mn, <S2>z1/2, [η] and Rh) of LRP, and transform the hyperbranched LRP molecules into flexible and extended chains, which would reaggregate to form spherical aggregates under long-time ultrasonication. Additionally, Congo red experiment combined with CD analysis indicated the existence of triple helix structure in LRP, which was still retained after ultrasonic treatment. Furthermore, under short-time ultrasonication, the spherical aggregates with some branched chains in the native LRP solution could disaggregate and form triple helixes that could be further arranged to a dense network structure, but the untangled LRP chains would reaggregate after long-time ultrasonication. Chemical compounds studied in this articleCongo red (PubChem CID: 11313); Sodium hydroxide (PubChem CID: 14798); Potassium bromide (PubChem CID: 253877).