Apple polyphenol oxidase (PPO), a key enzyme triggering enzymatic browning in fruits and vegetables, requires efficient purification for both mechanistic studies and industrial applications. To address the limitations of conventional single precipitants (e.g., high solvent consumption and low enzyme activity recovery), this study aimed to develop a cost-effective PPO purification strategy by integrating mixed solvent precipitation guided by molecular dynamics (MD) simulations with chromatographic techniques. First, MD simulations predicted the interaction mechanisms between PPO and precipitants (ammonium sulfate, ethanol, acetone, and ethanol/acetone mixtures). Further purification was then conducted using DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephadex G-100 gel filtration. The experimental validation demonstrated that the ethanol/acetone mixed solvent precipitant exhibited superior binding affinity. Single-step mixed solvent precipitation (ratio of ethanol/acetone 1:1, v/v) at 50% solvent concentration reduced precipitant usage by 33.33%, while achieving 89.85% enzyme activity recovery and a 1.24-fold purification. After chromatography, the PPO specific activity reached 156362.78 U/mg, with a purification fold of 18.33. SDS-PAGE analysis confirmed a single protein band at approximately 28 kDa. The mixed solvent precipitation approach proposed in this study based on MD provides a novel approach for PPO purification, offering critical implications for advancing enzymatic browning control technologies in fruit and vegetable processing industries.