Paeonol, an important constituent of the traditional Chinese medicine Cortex Moutan, has a variety of bioactivity. A simple and sensitive HPLC–UV method has been developed for analysis of paeonol in different rat tissue (heart, liver, spleen, lung, kidney, and brain). Bio-samples were prepared by simple protein precipitation, and separation of paeonol was achieved on a C18 column with methanol–2% glacial acetic acid solution 70:30 (v/v) as mobile phase at a flow rate of 1.0 mL min−1. UV detection was at 274 nm and the column temperature was 30 °C. Linearity was good between 0.025 and 5.0 μg mL−1 (r 2 ≥ 0.9990) for tissue samples. Inter-day and intra-day accuracy (as relative error, RE) and precision (as relative standard deviation, RSD) were <5.94 and 6.05%, respectively. The limit of detection was 0.025 μg mL−1 and extraction recovery for all samples was ≥85.86%. The method was successfully applied to a tissue-distribution study after oral administration of 40 mg kg−1 paeonol to healthy Sprague–Dawley rats. The study showed that paeonol was quickly distributed and eliminated after oral administration; liver and kidney were the major distribution tissues of paeonol in rats, and paeonol quickly passed through the blood–brain barrier. It was also found there was no long-term accumulation of paeonol in rat tissues.