A label-free fluorescence aptasensor for the determination of 17β-estradiol (E2) was developed. The aptasensor was prepared by the functionalization of a diamond surface using a nitrogen radical beam (NRB) system to produce amine terminals on the diamond itself. The fabrication of the sensor was performed by photolithography to produce a dot pattern, which was used as a sensing area for activated biomolecules. The supporting DNA strands were immobilized and an aptamer was hybridized to prepare a detection pair to bind with any E2 molecule, as the aptamer captures the E2 molecule naturally. The fluorescence microscopy signal was used for the detection of E2. The biomolecule activities were determined through special treatment, with three different stages evident in the fluorescence result, namely, hybridization, detection, and denaturation. When E2 was detected by the aptamer, the intensity of the fluorescence signal decreased because of the aptamer binding with the E2 molecule. E2 was detected by determining the fluorescence signal intensity of the dot pattern. When the aptamer was released from the supporting DNA and formed a complex with the E2 molecule, the intensity of the dot pattern decreased rapidly. The difference between the two signals indicated the number of E2 molecules bound to the aptamer. The proposed method is simple and powerful for the detection of small molecules with high sensitivity by utilizing the basic reaction of aptamers.