Exosomes are small membrane bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. Currently, a standard method for serum exosome isolation is differential ultracentrifugation, but a search for alternative, less time-consuming and labour extensive exosomal isolation method for use in clinical settings is ongoing. The effect of serum exosome isolation method on obtained miRNA profile is not yet clear. The aim of this study was to determine to which extent selected exosome isolation methods influence the serum exosomal miRNA profile. Exosomes were isolated from blood serum of healthy individuals by ultracentrifugation and ExoQuick Precipitation methods. The expression profile of 375 miRNAs was determined by real time PCR using Exiqon miRCURY LNA™ microRNA Human panel I assays. Although a strong correlation of exosomal miRNA profiles was observed between the two isolation methods, distinct clusters of miRNA levels between the used methods were identified. The detected levels of two miRNAs, miR-92a and miR-486-5p, were significantly influenced by the exosome isolation method used. Both exosome isolation methods are suitable for serum exosomal miRNA profiling. Differences found in miRNA patterns between the two methods indicate that the observed exosomal miRNA profile is slightly affected by the extracellular vesicle isolation method. •miRNA profiles obtained by two exosomal isolation methods were compared.•Used exosome isolation methods are suitable for serum exosomal miRNA profiling.•Exosomal miRNA profile is somewhat dependent upon the exosome isolation method.•Higher miRNA yield in serum exosomes was detected by ExoQuick method.