A high level of folate receptor (FR) expression is found frequently in numerous human cancerous cells, and monitoring of the FRs level is important for the diagnosis and treatment of cancers and chronic inflammatory diseases. In this study, a simple but sensitive fluorescent biosensor for FR detection in cancer cells has been proposed, which combines the desirable specificity of terminal protection and high amplification efficiency of hyperbranched rolling circle amplification (HRCA). Single-stranded DNA (ssDNA) terminally tethered to folic acid (FA) can specifically bind with FR, and therefore be protected from degradation by exonuclease I (Exo I). The protected ssDNA can hybridize with the padlock probe and initiate the HRCA reaction. In contrast, ssDNA will be degraded by Exo I and no probe is left to trigger the HRCA reaction without the protection of the target FR. The products of HRCA contain a large amount of double-strand DNA (dsDNA) and a strong fluorescence signal can be detected after the addition of SYBR Green I. The enhanced fluorescence intensity has a linear relationship with the logarithm of FR (standard solution) concentration ranging from 0.18 fM to 22 pM with a detection limit of 0.06 fM. Moreover, the proposed biosensor has been applied to detect FR in cancer cells (HeLa cells as an example). The enhanced fluorescence intensity has a linear dependence on the logarithm of cell concentration in the range of 50–5000 cell per mL. This biosensor has potential applications in the area of early cancer diagnosis.