Insight into the structure and conformation characteristics of starch that influence its enzyme susceptibility is import for its potential application. In this study, the capacity of asymmetrical flow field-flow fractionation (AF4) coupled online with multi-angle light scatting (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI) for monitoring of change in structure and conformation of potato starch during enzymatic hydrolysis was evaluated. The dissolution behavior of potato resistant starch (type 2) (PRS) was investigated. The effect of incubation time and amyloglucosidase concentration on the structure and conformation of potato starch was studied. The apparent density and the ratio of /2, (radius of gyration) to Rh (hydrodynamic radius) obtained from AF4-MALS-dRI were proven to be important parameters as they offer an insight into conformation of PRS at molecular level. Results suggested that gelatinization process made potato amylose molecules have a loose and random coil conformation which could contribute to an acceleration of enzymatic hydrolysis of potato starch. Furthermore, an intermediate with an elongated branched conformation was found between amylose and amylopectin populations, which may play a role in digestion property of potato starch. The results demonstrated that AF4-MALS-dRI is a powerful tool for better understanding of conformation of PRS.