The work aims to explore the source of acetaldehyde in polyester eye drop bottle and establish a method for the determination of acetaldehyde. The acetaldehyde content in polyester eye drop bottles placed at room temperature for 0 and 90 days was determined by optimizing acetaldehyde determination method. The change of acetaldehyde content was observed, and the migration of acetaldehyde into the eye drop bottles at room temperature was investigated. The acetaldehyde content in the eye drop bottle decreased significantly after 90 days, and the acetaldehyde migrated to the eye drop bottle. The change value of acetaldehyde content and the migration amount after 1 day at room temperature were much larger than the residual acetaldehyde content, which proved that the polyester bottle could react to produce acetaldehyde at room temperature. Strong linearity was shown, when the acetaldehyde injection volume was 0.2-5 μg, the sample precision RSD was 2.3%, the average recoveries were 85.2%, 87.9% and 89.6% from low to high, respectively, indicating that the optimized method could accurately quantify acetaldehyde. The source of acetaldehyde has been studied, and it is determined that polyester eye drop bottle can produce acetaldehyde at normal temperature. The optimized method can determine the acetaldehyde content in polyester eye drop bottle quickly and accurately, and provide guidance for the production and quality control of polyester eye drop bottle.