Artificial membraneless organelle with molecularly crowded and confined microenvironment shows great potential for improving functional RNA properties and developing biosensing. However, the performance of current artificial membraneless organelles is impaired with polycationic components. In this work, we demonstrate the construction of a purely fluorescent RNA aptamer based artificial membraneless organelle (FRAME) for improving RNA performance and biosensing. The fluorescent RNA aptamer in FRAME exhibited higher enzymatic resistance, thermostability, photostability and binding affinity than that dispersed in solution. The FRAME can be easily designed as a biosensor for tetracycline detection in lake water and tap water, which eliminated the adverse effects of polycationic components and demonstrated about 42.8-fold sensitivity improvement than the biosensor prepared with Poly-L-Lysine and RNA. By introducing epithelial cell adhesion molecule (EpCAM) aptamer to specific recognition of tumor cells, the FRAME is easily engineered as an ultra-sensitive probe to precisely identify cancer cell phenotypes. We believe that the proposed FRAME system would provide a universal platform for biosensing.