AbstractAn improved optimization method was used, combining a single-factor experiment and Response Surface Methodology to optimize the medium for the extraction yield of polyprenols from the needles of Cunninghamia lanceolata (Lamb.) Hook . A three-factor, three-level Box-Behnken design was used with extraction temperatures, extraction times, and liquid-solid ratio as independent variables to understand and optimize the extraction yield of polyprenols. A mathematical model with a high coefficient of determination was obtained and could be employed to optimize polyprenols extraction. From the optimized values of extraction temperature 71.4 °C, extraction time 5.96 h, and liquid-solid ratio 9.3:1, the extraction yield of polyprenols was 1.22 ± 0.04% (N = 3), which agreed closely with the predicted value (1.27%). Besides, polyprenols were demonstrated to have a strong antioxidative ability in vitro. Scavenging percentages of superoxide radical and DPPH by polyprenols were up to 75.6% and 56.9%, respectively.