We describe herein a method for isolating fully-modified 4'-thioRNA aptamers against human alpha-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4'-thioribonucleoside triphosphates (4'-thioNTPs) in an effort to afford the fully-modified 4'-thioRNA. The transcription efficiency of the 4'-thioNTPs was first compared with that of the nucleoside 5'-(alpha-thio)triphosphates (alphaSNTPs) and found to be less effective than that of the alphaSNTPs, especially when GTP and/or ATP were substituted for 4'-thioGTP and/or 4'-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4'-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4'-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4'-thioRNA, but not a fully-modified 4'-thioRNA, we eventually succeeded in isolating the fully-modified 4'-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers.