In order to prepare the polyol with all bio-based components as raw materials, cottonseed oil was first epoxidized by peroxyformic acid generated in situ from hydrogen peroxide and formic acid, and the cottonseed oil based polyols with variable hydroxyl value were then prepared by the ring-opening of epoxidized cottonseed oil with sorbitol, which is a multi-functional hydroxyl compound derived from a natural source. The chemical structure of the products was characterized with FTIR analysis, and the residual epoxy oxygen content and hydroxyl value of the polyol versus the ring-opening time were investigated.