Abstract: The SELEX technology was used to select aptamers (Apt) of permeability glycoprotein (P-gp). The enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods were used to determine the affinity between Apts and P-gp. Apt5 was chosen to evaluate the inhibitory effect on P-gp efflux function with immortalized human brain capillary endothelial cells (hCMEC/D3) and human epithelial colorectal adenocarcinoma cells (Caco-2). As a result, ten of 81bp Apts were selected. The affinity order of Apt was Apt5>Apt8>Apt1>Apt4>Apt6>Apt10>Apt7>Apt2>Apt9>Apt3. Further research showed that Apt5 significantly increased the cellular accumulation of rhodamine 123 both in hCMEC/D3 and Caco-2 cells, with a fold of 40.77% (P<0.01) and 32.39% (P<0.05), respectively, which indicated that Apt5potentially inhibited the efflux function of P-gp. In present study, several Apt of P-gp are presented, which had high affinity with P-gp and could inhibit P-gp efflux function. These Apts might be potential novel P-gp inhibitor.