The invention belongs to the technical field of biology, and discloses an aptamer sensor for detecting beta-lactamase nucleic acid in dairy products and a preparation method and application of the aptamer sensor. The aptamer sensor is prepared through the following method that a GO solution is prepared; beta-lactamase with gradient concentration is taken and mixed with a fluorescein FAM labeled beta-lactamase nucleic acid aptamer sensor, incubation is conducted for 20 min under the condition that the temperature is 40 DEG C and the pH is 7.2, the GO solution is added, incubation is conducted for 10 min, and the fluorescence intensity is determined; a standard linear curve of the aptamer sensor is determined. On the basis of interaction of graphene oxide and aptamer, the aptamer sensor which is direct, rapid, sensitive and high in specificity is established, and beta-lactamase illegally added in the milk products on the market can be rapidly detected by means of the sensor. The important practical significance in fighting against illegal and criminal acts in the food field and ensuring food safety and health of people is achieved.