In this work, pyrolysis oil produced by the slow pyrolysis of pinewood was the feed solution for a freeze concentration study and cooling crystallization recovery of levoglucosan. Liquid-liquid extraction of water-soluble compounds was performed by adding water to the original pyrolysis oil with ratios of 1:1 and 1:5 water: pyrolysis oil (wt%), respectively. A two-step suspension freeze concentration was then performed with the 1:1 and 1:5 aqueous pyrolysis oils (AqPO) to concentrate the solutions. The efficiency of water removal from AqPO was assessed by measuring the ice yield and distribution coefficient of ice impurities. Levoglucosan recovery from a supersaturated 1:1 AqPO-water mixture was performed for 37.5 min and 60 min by cooling crystallization with an aging time of 60 min. The average recovery rate of levoglucosan was 24 wt% with mean levoglucosan crystal size of 831 μm.