Chaotropes could significantly enhance lipase activity recovery in reverse micellar back extraction. However, the mechanism of chaotropes promoting the release of enzyme from reverse micelles was not clear. In this study, chaotropes were added in the process of lipase reverse micellar extraction, and back extraction recovery was improved. In back extraction, at 0.6 M urea in stripping solution, 94.60 % total extraction recovery was obtained. Meanwhile at 0.3 M guanidine hydrochloride, nearly 65 % lipases were released into the stripping solution. DLS and Karl Fischer method results showed that the presence of urea in stripping solution could weaken the electrostatic interaction between lipase and cetyltrimethylammonium bromide molecules, promoting lipase release from reverse micelles, and guanidine hydrochloride could stimulate lipase and free water molecules enwrapped in reverse micelle release into the stripping solution. These experimental results provide a clue for understanding the mechanism of chaotropes influencing on protein recovery in reverse micelle back extraction.