Toll-like receptor 3 (TLR3) detects double-stranded RNA (dsRNA), known to be a universal viral molecular pattern, and activates the antiviral immune response. While TLR3 preferentially recognizes polyinosinic-polycytidylic acid (poly(I:C)), no sequence-specific dsRNA has been shown to activate TLR3. To determine whether TLR3 preferentially recognizes a specific RNA sequence or structure that acts upon the TLR3 signaling pathway, in vitro selection against the human TLR3 ectodomain (TLR3 ECD) was performed. After the seventh selection cycle, two major classes, Family-I and Family-II, emerged from 64 clones with binding constants of about 3 nM. To examine the structure–function relationship of Family-I and -II aptamers, mutational analyses and RNase mapping were carried out. Furthermore, to elucidate the effect of selected aptamers on TLR3 signaling in vivo, a reporter gene assay was conducted in cells. These aptamers did not have any agonistic or antagonistic effects on TLR3 signaling in TLR3-transfected HEK293 cells, although they bound to TLR3 ECD with high affinity in vitro. These results suggest that selection of RNA aptamers for TLR3 ECD should be performed under physiological conditions, since TLR3 ECD localizes in acidic compartments such as endosomes.