We develop for the first time a label-free and self-circulated fluorescent biosensor for sensitive detection of ten-eleven translocation 1 (TET1) activity in cancer cells. This biosensor displays good specificity and high sensitivity without the need for any antibodies/exogenous primers and it can accurately quantify TET1 activity at the single-cell level. Moreover, it can be employed for the measurement of kinetic parameters and screening of inhibitors.