One fluorescent aptasensor based on the fluorescence resonance energy transfer was constructed and used to detect insulin and dopamine. Aptamer labeled FAM (aptamer-FAM) was in charge of energy donor, while graphene oxide served as an energy acceptor. The results show that the linear detection range of insulin is from 0.05 μmol/L to 10 μmol/L and dopamine is from 1 μmol/L to 500 μmol/L, meanwhile the signal intensity of insulin is stronger than dopamine at the same concentration. The specificity experiments for insulin and dopamine have indicated that this fluorescent aptasensor have a good specificity. Due to its detection for different targets and high selectivity, this fluorescent aptasensor offers a promising prospect in the detection field of biological and medical.