The invention belongs to the field of detection of small molecular substances, in particular to a method for simultaneously detecting multiple aminoglycoside antibiotics. According to the method, a nitrocellulose membrane is utilized to solidify aminoglycoside antibiotic dox-ova, enzyme labeling is carried out on the aminoglycoside antibiotics, the aminoglycoside antibiotics is solidified on the membrane through specific binding of antigen-antibody, horse radish peroxidase or alkaline phosphatase reacts with a substrate to generate a product with a certain color, the product can exist stably for a long time, therefore, detection results can be directly read by naked eyes within 5-20 minutes; the rapid and simultaneous detection of multiple aminoglycoside antibiotics in a to-be-tested sample is achieved. Compared with a traditional immunoassay method, the method for simultaneously detecting the multiple aminoglycoside antibiotics has the advantages that the detection is efficient, the cost is low, and the time consumption is short; meanwhile, the detection results can be read by naked eyes, the method has convenience and practicability, is wider in application range, and is especially suitable for rapid screening of aminoglycoside antibiotics in liquid samples.