Agaro-oligosaccharides were hydrolytically obtained from agar using hydrochloric acid, citric acid, and cationic exchange resin (solid acid). The FT-IR and NMR data showed that the hydrolysate has the structure of agaro-oligomers. Orthogonal matrix method was applied to optimize the preparation conditions based on α-naphthylamine end-labeled HPLC analysis method. The optimal way for oligosaccharides with different degree of polymerization (DP) was achieved by using solid acid degradation, which could give high yield and avoid solution neutralization process. Agaro-oligosaccharides with high purity were consequently obtained by activated carbon column isolation. Furthermore, the antioxidant and α-glucosidase inhibitory activity of three fractions were also investigated. The result indicated that 8% ethanol-eluted fraction showed highest activity againstα-glucosidase with IC50 of 8.84mg/mL, while 25% ethanol-eluted fraction possessed excellent antioxidant ability.