The present paper aims to investigate the optimum amount of serum samples used to isolate exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit. Exosomes were isolated by Qiagen exoRNeasy Serum/Plasma Midi Kit from different gradient serum samples, including 250 μL, 500 μL, 1 000 μL. The particle size and morphology of exosomes were measured by transmission electron microscopy. The expressions of exosomal surface protein markers CD63 and TSG101 were determined by Western blotting. The expression of microRNA-122 (miR-122) in exosomes was detected by real-time fluorescent quantitative polymerase chain reaction (PCR). Serum exosomes were circular or elliptic under the transmission electron microscope with diameters of 30-150 nm and they had intact membrane structure. Western blotting results showed that CD63 and TSG101 were positive in exosomes. The real-time fluorescent quantitative PCR results showed that when the initial serum volume was 250 μL, 500 μL and 1 000 μL, the expression of miR-122 in chronic hepatitis B patients was up-regulated by 22.44 folds, 21.48 folds and 20.69 folds, respectively compared with controls (P＝0.42). It is suggested that a small amount of 250 μL serum could be used for isolation of exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit, when the clinical serum sample is limited.