Steroid estrogens, including 17β-estradiol (TE2), estrone (TE1), and ethinyl estradiol (TEE2), which are the strongest endocrine disruptors and coexist in the environment, seriously harm the health of organisms; thus, the monitoring of total steroid estrogens (TEs) has attracted growing attention. Herein, a method based on surfaceenhanced Raman spectroscopy (SERS) group-targeting detection is established to detect TEs in natural water for the first time. The TEs response detection range and detection limit were 0.01−50 nM and 5 pM, respectively. An antiinterference ability was observed: even if coexisting interfering species were present in the system at 100-fold the concentration of estrogens, the detection error of the method was less than 0.276. In addition, the association constants between the aptamers and TE1, TE2, and TEE2 were similar, and therefore, the recognition of TE1, TE2, and TEE2 by the aptamers was consistent. Furthermore, the interaction sites A44, T72, and G69 between the aptamers and TE1, TE2, and TEE2 were investigated by molecular docking. On this basis, the estrogens in environmental water samples, including animal farm wastewater, maternity hospital wastewater, surface water from near an animal farm, and surface water from near a maternity hospital, were successfully determined. E strogens, a kind of environmental endocrine disruptor, are