© 2014 American Chemical Society. Circulating microRNAs (miRNAs) are potential biomarkers useful in cancer diagnosis. They have been found to be bound to various carriers like proteins, lipoprotein particles, and exosomes. It is likely that only miRNAs in particular carriers, but not the overall quantity, are directly related to cancer development. Herein, we developed a method for rapid separation of different miRNA carriers in serum using asymmetrical flow field flow fractionation (AF4). Sera from two healthy individuals (control) or from two cancer patients (case) were fractionated. Six fractions enriching different types of miRNA carriers, such as the lipoprotein particles and exosomes, were collected. The quantities of eight selected miRNAs in each fraction were obtained by RT-qPCR to yield their distribution profiles among the carriers. Larger changes in miRNA quantity between the control and the case were detected in the fractionated results compared to the sum values. Statistical analysis on the distribution profiles also proved that, the quantities of 4 miRNAs within particular fractions showed significant difference between the controls and the cases. On the contrary, if the overall quantity of the miRNA was subject to the same statistical analysis, only 2 miRNAs exhibited significant difference. Moreover, principle component analysis revealed good separation between the controls and the cases with the fractionated miRNA amounts. All in all, we have demonstrated that, our method enables comprehensive screening of the distribution of circulating miRNAs in the carriers. The obtained distribution profile enlarges the miRNA expression difference between healthy individuals and cancer patients, facilitating the discovery of specific miRNA biomarkers for cancer diagnosis.