Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The light-up RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.