The photocatalytic fuel cell (PFC) provides an elegant approach for the construction of a light-induced self-powered sensing platform. Nevertheless, the quantification of a target with a single output signal in an ordinary PFC-based sensor is easily affected by variation of the light intensity and other environmental factors. Herein we propose a ratiometric self-powered aptasensor for highly selective detection of 17β-estradiol (E2) based on a dual-channel PFC constructed with two photoanodes which could effectively avoid the fluctuation of the light intensity. Taking the advantage of the inhibited output performance of PFC by E2 which was captured by aptamer immobilized on the photoanodes, E2 was quantified via the ratio of output power density values from dual photoanodes. Under optimum conditions, the ratiometric self-powered sensing signal was linearly related to the logarithm of E2 concentration in the range of 1 to 500 nM, with a detection limit (3S/N) of 0.12 nM. Thus, a new type of self-powered aptasensor with high accuracy and specificity was successfully developed based on PFC in cooperation with a ratiometric assay and a spatial-resolution technique.