A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.