Biosurfactant, produced by Pseudomonas aeruginosa BSZ-07, was added to the rice straw decomposing process to enhance the production of reducing sugars. Observed by Fourier Transform InfraRed (FT-IR) and Nuclear Magnetic Resonance [G2](NMR) analysis, the purified biosurfactant was considered as a mixture of RL1 and RL2, which are two different types of rhamnolipids. Two different adding methods, adding the purified rhamnolipid and the on-site production of it were compared. The results showed that 0.5 g/L was the optimum concentration for adding purified rhamnolipid and the optimum temperature for on-site production was 30℃ for the first 48 h and 34℃ for the next 48 h. Under the optimum conditions, these two adding methods could improve the production of reducing sugar to 2.730 g/L and 2.504 g/L, which was 22.30% and 12.20% higher than that of the rhamnolipid-free sample, respectively, which indicated that both of them were more effective than any other kind of surfactant discussed in this article. As the on-site production of rhamnolipid could omit the purification process, thus reducing the production cost effectively, it seemed to be a prospective adding method of the biosurfactant for enhancing rice straw decomposing.