A sensitive gene expression detection system is a valuable prognostic tool for detecting fatal diseases at early stage. Here a stem-loop structure aptamer based probe was used to develop an ELISA type assay for gene expression detection in vitro. Targeted Reversibly Activated Probe (TRAP) consists of a signaling module, aptamer, a short strand of nucleic acid complementary to part of the aptamer as attenuator and an intervening sequence which is complementary to a specific nucleic acid. Based on hybridization with the complementary nucleic acid, aptamer will be revealed from attenuator and bind to the signaling target. Previously developed ATP/DNA TRAP by our group was applied here for developing the ELISA type assay. However, after immobilizing the TRAP on different type of surfaces, it did not show the same binding activity as it showed with affinity column assay. It was speculated that combination of washing steps and rapid off rate of aptamer-target might be causing the problem. To overcome this problem, a new TRAP using thrombin aptamer was developed which allows creating covalent binding by UV treatment. The DNA/thrombin TRAP showed higher binding activity in the presence of its complementary nucleic acid. It also differentiated non-complementary nucleic acid having even one base difference with its complementary. However, due to a high ratio of noise to signal all these values become less significant. Binding activity of TRAP without regulatory DNA reduced along with the increase in the [Delta]G of folding. Also, due to higher stability, TRAPs with higher [Delta]G of folding were more difficult to be opened up by regulatory DNA. Two studies done here showed that a longer loop without secondary structure in it decreased the attenuation of the aptamer in closed state and increased its binding activity in the absence of regulatory DNA. We hypothesized that a longer loop without secondary structure keeps the aptamer and attenuator at far distance from each other, which makes the hybridization between them and shielding of the aptamer activity less likely. Characterization of TRAP's properties, done partly in this study, can provide a better understanding of TRAP activity which is helpful for designing different TRAPs.